Combination of immunotherapy with local chemotherapy for the treatment of malignancies

ABSTRACT

The presently disclosed subject matter provides methods, compositions, and kits for the treatment of cancer using a combination treatment comprising a locally administered chemotherapy and an immunotherapeutic agent. The presently disclosed subject matter also provides methods of promoting the combination treatment and instructing a patient to receive the combination treatment are also provided, as well immunotherapeutic, non-immunosuppressive compositions comprising the combination treatment, and methods of using the immunotherapeutic, non-immunosuppressive compositions for treating cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/151,619, filed Apr. 23, 2015, the contents of which are incorporatedherein by reference in their entirety.

BACKGROUND

Glioblastoma is the most common and aggressive primary brain tumor inadults. Despite multimodal treatment, clinical outcomes remain poor withthe majority of patients surviving less than 2 years (Stupp et al.,2009; Malmstrom et al., 2012; Gilbert et al., 2013). Althoughglioblastoma rarely metastasizes, these tumors and their treatment havesystemic sequelae, including severe immunosuppression (Parsa et al.,2007; Bloch et al., 2013; Brooks et al., 1972; Grossman et al., 2011).In an attempt to mitigate the systemic effects of chemotherapy and tomaximize the dose delivered directly to tumor cells, local intra-tumoralchemotherapy in the form of biodegradable BCNU wafers (Gliadel™ wafers,LC) was introduced in the early 1990s (Brem, 1990) and subsequentlyapproved by the FDA for recurrent GBM in 1996 (Brem et al., 1995) andfor newly diagnosed GBM in 2003 (Westphal et al., 2003).

Immunotherapy is an exciting approach to treating cancer. Immunotherapyapproaches are showing great promise. As an example, anti-PD1 monoclonalantibodies (mAbs) have emerged as a promising therapeutic strategy asPD1 blockade has demonstrated activity against a number of solid tumorsand appears to be associated with robust antitumor immunity with reducedadverse events compared to another effective mAb, CTLA-4 blockade or tothe combination of anti-PD1 and anti-CTLA-4 (Topalian et al., 2012;Brahmer et al., 2012; Hamid et al., 2013). PD1 is expressed on themembrane of tumor infiltrating lymphocytes (TILs) and interacts with itsligands PD-L1 and PD-L2, which are expressed on both tumor cells andimmune cells. The PD1-PD-L1 interaction results in decreased survivaland proliferation of CD8+ T-cells, reduced cytokine production, andeventually T cell exhaustion. Blockade of PD1-PD-L1 interaction canrestore the function of T-cells releasing an anti-tumor immune response(Pardoll, 2012; Blackburn et al., 2009). While immunotherapy carriesgreat potential, questions remain about the sequence or role ofchemotherapy with immunotherapy. Current thinking in clinical trialdesign discourages the combination of chemotherapy and immunotherapy dueto the concern that the immunosuppressive side effects of chemotherapymay blunt the activity of immunotherapeutic agents (Nowak et al., 2002;Sarkaria et al., 2010; van der Most et al., 2005).

SUMMARY

The practice of the present invention will typically employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, transgenic biology, microbiology,recombinant nucleic acid (e.g., DNA) technology, immunology, and RNAinterference (RNAi) which are within the skill of the art. Non-limitingdescriptions of certain of these techniques are found in the followingpublications: Ausubel, F., et al., (eds.), Current Protocols inMolecular Biology, Current Protocols in Immunology, Current Protocols inProtein Science, and Current Protocols in Cell Biology, all John Wiley &Sons, N.Y., edition as of December 2008; Sambrook, Russell, andSambrook, Molecular Cloning. A Laboratory Manual, 3^(rd) ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. andLane, D., Antibodies—A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, 1988; Freshney, R. I., “Culture of AnimalCells, A Manual of Basic Technique”, 5th ed., John Wiley & Sons,Hoboken, N.J., 2005. Non-limiting information regarding therapeuticagents and human diseases is found in Goodman and Gilman's ThePharmacological Basis of Therapeutics, 11th Ed., McGraw Hill, 2005,Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw-Hill/Appleton& Lange 10^(th) ed. (2006) or 11th edition (July 2009). Non-limitinginformation regarding genes and genetic disorders is found in McKusick,V. A.: Mendelian Inheritance in Man. A Catalog of Human Genes andGenetic Disorders. Baltimore: Johns Hopkins University Press, 1998 (12thedition) or the more recent online database: Online MendelianInheritance in Man, OMIM™. McKusick-Nathans Institute of GeneticMedicine, Johns Hopkins University (Baltimore, Md.) and National Centerfor Biotechnology Information, National Library of Medicine (Bethesda,Md.), as of May 1, 2010, World Wide Web URL:http://www.ncbi.nlm.nih.gov/omim/ and in Online Mendelian Inheritance inAnimals (OMIA), a database of genes, inherited disorders and traits inanimal species (other than human and mouse), athttp://omia.angis.org.au/contact.shtml. The Kinetochore, Springer, 2009.All patents, patent applications, and other publications (e.g.,scientific articles, books, websites, and databases) mentioned hereinare incorporated by reference in their entirety. In case of a conflictbetween the specification and any of the incorporated references, thespecification (including any amendments thereof, which may be based onan incorporated reference), shall control. Standard art-acceptedmeanings of terms are used herein unless indicated otherwise. Standardabbreviations for various terms are used herein.

In one aspect, the presently disclosed subject matter provides a methodfor the treatment of cancer comprising administering to a patient with acancer an effective amount of a combination treatment comprising: (a) alocally administered chemotherapy; and (b) an immunotherapeutic agent.

In certain aspects, the presently disclosed subject matter provides amethod of promoting a combination treatment for the treatment of apatient with a cancer, wherein the combination treatment comprises: (a)a locally administered chemotherapy; and (b) an immunotherapeutic agent.

In some aspects, the presently disclosed subject matter provides amethod for prolonging survival of a cancer patient comprisingadministering to a patient with a cancer an effective amount of acombination treatment comprising: (a) a locally administeredchemotherapy; and (b) an immunotherapeutic agent.

In another aspect, the presently disclosed subject matter provides amethod of instructing a patient with a cancer by providing instructionsto receive a combination treatment comprising; (a) a locallyadministered chemotherapy; and (b) an immunotherapeutic agent, to extendsurvival of the patient.

In other aspects, the presently disclosed subject matter provides a kitcomprising: (a) a locally administered chemotherapy; (b) animmunotherapeutic agent; and (c) a package insert or label withdirections to treat a patient with a cancer by administering acombination treatment comprising the locally administered chemotherapyand the immunotherapeutic agent.

In certain aspects, the presently disclosed subject matter provides animmunotherapeutic, non-immunosuppressive composition comprising: (a) alocally administered chemotherapy; and (b) an immunotherapeutic agent.

In other aspects, the presently disclosed subject matter relates to theuse of the immunotherapeutic, non-immunosuppressive composition for thetreatment of a cancer.

In still other aspects, the presently disclosed subject matter relatesto the use of the immunotherapeutic, non-immunosuppressive compositionfor the manufacture of a medicament for the treatment of a cancer.

Certain aspects of the presently disclosed subject matter having beenstated hereinabove, which are addressed in whole or in part by thepresently disclosed subject matter, other aspects will become evident asthe description proceeds when taken in connection with the accompanyingExamples and Figures as best described herein below.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus described the presently disclosed subject matter in generalterms, reference will now be made to the accompanying Figures, which arenot necessarily drawn to scale, and wherein:

FIG. 1 shows a representative experimental schedule of chemotherapy.Systemic chemotherapy shows severe and persistent lymphodepletion whilelocal chemotherapy (LC) maintains the immune cell populations intact.Mice were treated with either i.p. BCNU or LC at day 14. i.p. BCNU wasgiven 3 times a week for 2 weeks. Flow analysis was performed for allgroups for lymphocytes extracted from the brain, draining lymph nodesand the peripheral blood at several time points;

FIG. 2 shows early (day 26) and late (day 41) lymphopenia observed inthe peripheral blood of mice treated with i.p. BCNU. The same patternwas observed in the brain and the draining lymph nodes (DLN) of micetreated with systemic chemotherapy. i.p. BCNU exhibited latemyelotoxicity (day 30) as indicated by the reduced bone marrowcellularity and the reduced CD45+(leukocytes) population. EP=EmptyPolymer (no chemotherapy);

FIG. 3 shows CD3 counts at day 141. Persistent and potentiallyirreversible lymphopenia is observed in mice receiving i.p. BCNU. Thelymphocyte counts (CD3+ cells) were decreased in the peripheral blood,the draining lymph nodes and the spleen of long term survivor micetreated with i.p. BCNU compared to LC treated mice or untreated(control) mice;

FIG. 4 shows that the use of local chemotherapy post anti-PD1immunotherapy exhibits a superior survival profile compared tomonotherapies as well as the combination of systemic chemotherapy andanti-PD1 treatment. The experimental set-up is shown (top);

FIG. 5 shows the tumor progression of intracranially implanted GL-261tumors measured by bioluminescent imaging. Anti-PD1 treatment exhibitsan immediate anti-tumor effect with most mice that are finally curedlosing their bioluminescent signal at day 14. Interestingly, some micetreated with anti-PD1 and i.p. BCNU initially lost their signal but theyeventually regained it and died of a progressing tumor. *Denotes micethat died after the last IVIS imaging session;

FIG. 6 shows that the combination of LC and anti-PD1 mounts a robustlocal and systemic immune response, while systemic chemotherapy incombination with anti-PD1 abrogates the immune activation anti-PD1 alonecreates. Systemic chemotherapy in combination with anti-PD1 exhibitslymphopenia in the peripheral blood while LC and anti-PD1 maintains highlymphocyte counts at all time points;

FIG. 7 shows an increased number of CD3+ peripheral blood lymphocytes(PBLs) compared to control mice for a prolonged period of time (day 41)after the end of the therapeutic regimen;

FIG. 8 shows that systemic chemotherapy in combination with anti-PD1exhibits decreased numbers of CD8+ IFNγ producing cells (effector cells)compared to LC and anti-PD1 in the DLNs. A decreased percentage ofCD4+Foxp3+(Tregs) cells was exhibited with both systemic chemotherapyand anti-PD1 as well as LC and anti-PD1;

FIG. 9 shows that systemic chemotherapy in combination with anti-PD1exhibits lymphopenia in the DLNs while LC and anti-PD1 maintains highlymphocyte counts. The percent of Tregs is decreased with any treatmentand the percent of CD8+ IFNγ+(Teffectors) cells was highest in thecombination of LC and anti-PD1;

FIG. 10 shows that bone marrow cellularity was decreased in mice treatedwith systemic chemotherapy and anti-PD1. Gating on CD45+ cells(leukocytes) reveals decreased leukocyte production from the bone marrowin mice treated with systemic chemotherapy or systemic chemotherapy andanti-PD1;

FIG. 11 shows that analysis of specific myeloid subpopulations in thebone marrow (BM) reveals an increased granulocytic population(CD11b+Ly6G+Ly6C(intermediate) in the bone marrow of mice treated withsystemic chemotherapy and anti-PD1 and a decreased monocytic population(CD11b+Ly6G-Ly6Chigh) compared to any other treatment. Anti-PD1 or LCdid not preferentially affect a specific subpopulation of myeloid cells;

FIG. 12 shows flow cytometric analysis of bone marrow granulocytic andmonocytic myeloid-derived suppressor cells (MDSCs);

FIG. 13 shows that analysis of tumor infiltrating lymphocytes showslymphodepletion in the systemic chemotherapy groups and an immunesuppressive phenotype. At day 21, LC and anti-PD1 exhibits high numbersof TILs and increased percentage of Teffectors compared to i.p.BCNU+anti-PD1 or LC alone. At day 30, systemic chemotherapy groupsexhibited lymphodepletion and decreased Teffector function (decreasedIFNγ production). However, LC increased the Teffector function resultingin an increased effector T cell (Teff)/regulatory T cell (Treg) ratio;

FIG. 14 shows that the addition of anti-PD1 antibody to LC treatmentincreased the percentage of CD8-IFNγ producing cells;

FIG. 15 shows that the addition of anti-PD1 to LC treatment did notsignificantly decrease the percentage of regulatory T cells (Tregcells);

FIG. 16 shows the analysis of tumor infiltrating immune cells in tumorbearing mice for all treatment groups. CD11b+CD45 intermediate cells aremicroglial cells, CD11b+CD45high are myeloid cells and CD11b-CD45 highare tumor infiltrating lymphocytes (TILs);

FIG. 17 shows that the anti-PD1 treated group is enriched for TILscompared to microglia-macrophages at both Day 21 and 30. However, noneof the other treatment groups showed any changes in the TILs/Myeloidcells ratio;

FIG. 18 shows gating on the CD45+ cells (leukocytes) infiltrating thebrain. Focusing on the CD11b+CD11c+ cells (dendritic cells), anincreased infiltration of dendritic cells in the LC treated mice wasobserved;

FIG. 19 shows the use of chemotherapy prior to immunotherapy—survivalanalysis: LC and anti-PD1 exhibits an increase in survival compared tomonotherapies or i.p. BCNU and anti-PD1. The combination of i.p. BCNUwith anti-PD1 abrogates the positive survival profile anti-PD1 producedas monotherapy;

FIG. 20 shows the adoptive transfer of OT-I lymphocytes from RAG−/− micein GL-261 ova tumor bearing mice. LC allows for expansion of antigenspecific T cells intratumorally and in the DLNs. Adoptively transferredOT-I lymphocytes expressing the congenic marker CD45.2 were recoveredfrom recipient mice 4 days after the transfer; LC increased the homingand expansion of OT-I cells in the LC and LC+anti-PD1 groups compared toanti-PD1 or i.p. BCNU and i.p. BCNU+anti-PD1;

FIG. 21 shows adoptive transfer of OT-I lymphocytes from RAG−/− mice inGL-261 ova tumor bearing mice;

FIG. 22 shows in vivo proliferation of adoptively transferred OT-I Tcells in the spleen. Splenocytes from OT-I RAG−/− CD45.2 mice werelabeled with cell proliferation dye and were adoptively transferred toCD45.1 B6 mice treated with either i.p. BCNU or LC (and the appropriatecontrols: EP and No Tx). Three days after adoptive transfer, therecipient mice were sacrificed and their spleen was harvested. The invivo proliferation capacity of CD45.2+CD3+ transferred lymphocytes wasassessed by the dilution of cell proliferation dye;

FIG. 23 shows that that systemic chemotherapy abrogates the antitumormemory response in anti-PD1 treated mice and causes functionalimpairment of T memory cells. Long term survivor mice from all groupswere rechallenged; mice in the local chemotherapy groups and theanti-PD1 group prevented tumor recurrence while systemic chemotherapytreated mice failed to reject the rechallenged tumor and died;

FIG. 24 shows a representative study in which long term survivors werere-challenged with GL-261 cells implanted in the contralateralhemisphere of the brain while naïve mice with no previous exposure totumor cells were challenged in parallel. Rechallenged mice were followedwith bioluminescent imaging; mice treated with systemic chemotherapyexhibited progressively increasing bioluminescent imaging (BLI) signaland eventually died of their tumor. *Denotes mice that died after thelast IVIS imaging session. Naïve: GL-261 implanted, non-treated mice;anti-PD1 R: anti-PD1 rechallenged mice; BCNU R:BCNU rechallenged mice;i.p. BCNU+anti-PD1 R: i.p. BCNU+anti-PD1 Rechallenged mice; LC+anti-PD1R: local chemotherapy+anti-PD1 rechallenged mice; and anti-PD1: micewith primary tumor treated with anti-PD1 for the first time;

FIG. 25 shows a representative day 20 post re-challenge experiment. Thepercent of effector CD8 memory cells producing IFNγ was lower in thesystemic chemotherapy treated mice across all peripheral tissuesincluding the spleen, peripheral blood and DLNs;

FIG. 26 shows a representative flow cytometric analysis from a day 20post re-challenge experiment (FIG. 25). Twenty days after intracranialtumor re-challenge, long-term survivor mice were assessed for thepresence of memory cells;

FIG. 27 shows therapeutic adoptive transfer of CD8 cells fromrechallenged LC+anti-PD1 mice to i.p. BCNU+anti-PD1 rechallenged mice.Long term survivor mice from the LC+anti-PD1 group rejected therechallenged tumor whereas i.p. BCNU+anti-PD1 did not. Spleens from micetreated with LC+anti-PD1 R group were harvested and CD8 cells weremagnetically isolated. The cells were adoptively transferred viaretro-orbital injection to the rechallenged i.p. BCNU+anti-PD1 mice. Twoout of four mice (50%) did not respond to the transfer whereas the other50% (two mice) showed a considerable decrease in the tumor burden andmaintained a state of tumor equilibrium with steady bioluminescentsignal for more than a week;

FIG. 28 shows the adoptive transfer of CD8 cells from LC+anti-PD1 R miceto i.p. BCNU+anti-PD1 R mice. Bioluminescent imaging shows theprogression of the tumor for naïve mice and rechallenged long termsurvivor mice from the i.p. BCNU+anti-PD1 group with or without the CD8adoptive transfer. Two out of four mice in the i.p. BCNU+anti-PD1 R withCD8 AT (Adoptive Transfer) group died (denoted with asterisk) and twoout of four mice showed a definite decrease in bioluminescent signalafter the adoptive transfer and maintained similar signal for at least aweek from day 17 to day 24. *Denotes mice that died after the last IVISimaging session. Naïve: GL-261 implanted, non-treated mice; anti-PD1 R:anti-PD1 rechallenged mice; BCNU R:BCNU rechallenged mice; i.p.BCNU+anti-PD1 R: i.p. BCNU+anti-PD1 rechallenged mice; LC+anti-PD1 R:local chemotherapy+anti-PD1 rechallenged mice; anti-PD1: mice withprimary tumor treated with anti-PD1 for the first time;

FIG. 29 shows the anti-PD1 response to chemotherapy naïve andchemotherapy treated mice. Mice treated with systemic chemotherapy donot respond to anti-PD1 treatment as chemotherapy naïve mice. Mice withrecurrent tumor after tumor rechallenge were treated with anti-PD1 in anattempt to be cured. Chemotherapy naïve mice were treated with anti-PD1at the same time as controls with the mice developing recurrent tumorsnot responding to anti-PD1. Naïve: GL-261 implanted, non-treated mice;anti-PD1 R: anti-PD1 rechallenged mice; BCNU R:BCNU rechallenged mice;i.p. BCNU+anti-PD1 R: i.p. BCNU+anti-PD1 rechallenged mice; LC+anti-PD1R: local chemotherapy+anti-PD1 rechallenged mice; and anti-PD1: micewith primary tumor treated with anti-PD1 for the first time. *Denotesmice that died after the last IVIS imaging session;

FIG. 30 shows that the T effector memory function of rechallenged micein the systemic chemotherapy group cannot be restored by anti-PD1treatment. Naïve: GL-261 implanted, non-treated mice; anti-PD1 R:anti-PD1 rechallenged mice; BCNU R:BCNU rechallenged mice; i.p.BCNU+anti-PD1 R: i.p. BCNU+anti-PD1 rechallenged mice; LC+anti-PD1 R:local chemotherapy+anti-PD1 rechallenged mice; and anti-PD1: mice withprimary tumor treated with anti-PD1 for the first time;

FIG. 31 is a graph depicting a quantification of the results obtained inFIG. 30, demonstrating that the T effector memory function ofrechallenged mice in the systemic chemotherapy group cannot be restoredby anti-PD1 treatment as seen by flow cytometry plots in FIG. 30; and

FIG. 32 shows necropsy findings at Day 104. Upon necropsy, the spleensof mice treated with systemic chemotherapy and anti-PD1 or systemicchemotherapy and rescue anti-PD1 were much smaller than the spleens ofLC and anti-PD1 mice. Furthermore, mice treated with systemicchemotherapy and rechallenged on the opposite hemisphere (right) thanthe initial tumor implantation (left) exhibited big tumor massesindicating uncontrolled tumor progression, unlike the LC and anti-PD1rechallenged mice that did not show any sign of tumor recurrence. Naïve:GL-261 implanted, non-treated mice; anti-PD1 R: anti-PD1 rechallengedmice; BCNU R:BCNU rechallenged mice; i.p. BCNU+anti-PD1 R: i.p.BCNU+anti-PD1 rechallenged mice; LC+anti-PD1 R: localchemotherapy+anti-PD1 rechallenged mice; and anti-PD1: mice with primarytumor treated with anti-PD1 for the first time.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fullyhereinafter with reference to the accompanying Figures, in which some,but not all embodiments of the inventions are shown. Like numbers referto like elements throughout. The presently disclosed subject matter maybe embodied in many different forms and should not be construed aslimited to the embodiments set forth herein; rather, these embodimentsare provided so that this disclosure will satisfy applicable legalrequirements. Indeed, many modifications and other embodiments of thepresently disclosed subject matter set forth herein will come to mind toone skilled in the art to which the presently disclosed subject matterpertains having the benefit of the teachings presented in the foregoingdescriptions and the associated Figures. Therefore, it is to beunderstood that the presently disclosed subject matter is not to belimited to the specific embodiments disclosed and that modifications andother embodiments are intended to be included within the scope of theappended claims.

Systemic chemotherapy causes immunosuppression, which in turn abrogatesthe efficacy immune checkpoint molecules have against cancer. Thepresently disclosed subject matter provides methods comprising the useof local chemotherapy, such as intratumorally or within the tumor bed,to avoid the systemic immunosuppressive effects of systemic chemotherapycombined with at least one immunotherapeutic agent. In some embodiments,the local chemotherapy further enhances the antitumor activity of theimmunotherapeutic agent. In some embodiments, at least oneimmunotherapeutic agent is an immune checkpoint molecules. This novelcombination of local chemotherapy with an immunotherapeutic agent hasthe potential to change the way patients can be treated in a variety ofmalignancies.

As seen herein below, the combination of local chemotherapy and animmune checkpoint molecules, such as an anti-PD1 antibody, exhibits astrong survival and immunologic benefit compared to treatment withsystemic chemotherapy and an immune checkpoint molecules, or monotherapytreatment using local chemotherapy or the immune checkpoint moleculesalone. The presently disclosed subject matter demonstrates superiorefficacy of local chemotherapy delivery in combination withimmunotherapy for primary brain tumors (gliomas), such as glioblastomas.The presently disclosed subject matter can be used for treating manyother tumor types currently treated with systemic chemotherapy.

I. Methods for Treating Cancer

In some embodiments, the presently disclosed subject matter provides amethod for the treatment of cancer comprising administering to a patientwith a cancer an effective amount of a combination treatment comprising:(a) a locally administered chemotherapy; and (b) an immunotherapeuticagent. In some embodiments, the presently disclosed subject matterprovides a method for prolonging survival of a cancer patient comprisingadministering to a patient with a cancer an effective amount of acombination treatment comprising: (a) a locally administeredchemotherapy; and (b) an immunotherapeutic agent. In some embodiments,the immunotherapeutic agent comprises an immune checkpoint molecule. Insome embodiments, the locally administered chemotherapy is administeredintratumorally and/or within a tumor bed.

As used herein, the term “treating” can include reversing, alleviating,inhibiting the progression of, preventing or reducing the likelihood ofthe disease, disorder, or condition to which such term applies, or oneor more symptoms or manifestations of such disease, disorder orcondition (e.g., cancer).

In some embodiments, the combination treatment reduces the likelihood oftumor progression and/or recurrence. For example, the combinationtreatment can reduce the likelihood of tumor progression and/orrecurrence by at least 5%, 10%, 15%, 20%, 25%, 30%, 33%, 35%, 40%, 45%,50%, 55%, 60%, 66%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more as compared to the likelihood of tumorprogression and/or recurrence in the patient when treated with systemicchemotherapy plus the immunotherapeutic agent, such as an immunecheckpoint molecule, or as compared to monotherapy treatment with eitherthe locally administered chemotherapy or the immunotherapeutic agentalone. In some embodiments, the combination treatment completelyinhibits tumor progression and/or recurrence in the patient. In someembodiments, the combination treatment reduces the likelihood of tumorprogression and/or recurrence by at least approximately 40% as comparedto the likelihood of tumor progression and/or recurrence in the patientwhen treated with systemic chemotherapy plus the immunotherapeuticagent, or as compared to monotherapy treatment with either the locallyadministered chemotherapy or the immunotherapeutic agent alone.

In some embodiments, the combination treatment extends survival of thepatient. For example, the combination treatment can extend survival(e.g., progression free survival) of the patient by 5%, 10%, 15%, 20%,25%, 30%, 33%, 35%, 40%, 45%, 50%, 55%, 60%, 66%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 1-fold, 1.1-fold,1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold,1.9-fold, 2.0 fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 5.0-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more as compared to survivalof the patient when treated with systemic chemotherapy plus theimmunotherapeutic agent, or as compared to monotherapy treatment witheither the locally administered chemotherapy or the immunotherapeuticagent alone. In some embodiments, the combination treatment extendssurvival of the patient by at least approximately 40% as compared tosurvival of the patient when treated with systemic chemotherapy plus theimmunotherapeutic agent, or as compared to monotherapy treatment witheither the locally administered chemotherapy or the immunotherapeuticagent alone.

In some embodiments, the combination treatment extends progression freesurvival of the patient until the patient succumbs to another disease,disorder, or condition, or dies naturally as a result of old age.

In some embodiments, the combination treatment stimulates an anti-tumorresponse in the patient in the absence of inducing immunosuppression inthe patient. Unexpectedly and surprisingly, work described hereindemonstrates that combination treatment using local chemotherapy (e.g.,a locally administered chemotherapy administered intratumorally orwithin the tumor bed delivered, for example, by catheters, polymers, ornanoparticles) and an immunotherapeutic agent stimulated an anti-tumorimmunotherapeutic response, for example by increasing dendritic cellinfiltration in the tumor microenvironment, increasing the persistenceof antigen specific T cells specific for a tumor antigen of the patient,enhancing retention of immunologic memory upon recurrence of the tumoror tumor antigen, increasing clonal expansion of antigen specific Tcells, increasing antigen release from local chemotherapy induced tumorcell death, increasing and/or maintaining CD3+(lymphocyte) cell countsin the patient, increasing and/or maintaining CD45+(leukocyte) cellcounts in the patient, increasing and/or maintaining CD8+ IFNγ producingcell (effector cell) counts in the patient in the absence of inducingimmunosuppression in the patient, for example by stimulating theanti-tumor immunotherapeutic response noted above without causing thepatient to exhibit lymphopenia (reduced CD3+ count), myelotoxicity,reduced bone marrow cellularity, leukopenia (reduced CD45+ count),reduced CD8+ IFNγ producing cell (effector cell) count; functionalimpairment of T memory cells. A “cancer” in a patient refers to thepresence of cells possessing characteristics typical of cancer-causingcells, for example, uncontrolled proliferation, loss of specializedfunctions, immortality, significant metastatic potential, significantincrease in anti-apoptotic activity, rapid growth and proliferationrate, and certain characteristic morphology and cellular markers. Insome circumstances, cancer cells will be in the form of a tumor; suchcells may exist locally within an animal, or circulate in the bloodstream as independent cells, for example, leukemic cells. Cancer as usedherein includes newly diagnosed or recurrent cancers, including withoutlimitation, blastomas, carcinomas, gliomas, leukemias, lymphomas,melanomas, myeloma, and sarcomas. Cancer as used herein includes, but isnot limited to, head cancer, neck cancer, head and neck cancer, lungcancer, breast cancer, prostate cancer, colorectal cancer, esophagealcancer, stomach cancer, leukemia/lymphoma, uterine cancer, skin cancer,endocrine cancer, urinary cancer, pancreatic cancer, gastrointestinalcancer, ovarian cancer, cervical cancer, and adenomas. In someembodiments, the cancer comprises Stage 0 cancer. In some embodiments,the cancer comprises Stage I cancer. In some embodiments, the cancercomprises Stage II cancer. In some embodiments, the cancer comprisesStage III cancer. In some embodiments, the cancer comprises Stage IVcancer. In some embodiments, the cancer is refractory and/or metastatic.For example, the cancer may be refractory to treatment withradiotherapy, systemic chemotherapy, monotreatment with localchemotherapy, or monotreatment with immunotherapy. In some embodiments,the cancer is metastatic colorectal cancer. In some embodiments, thecancer is diffuse large B-cell lymphoma. In some embodiments, the canceris follicular lymphoma (e.g., refractory follicular lymphoma). In someembodiments, the cancer is glioblastoma. In some embodiments, the canceris multiple myeloma comprising solid tumors (see, e.g., Stegman andAlexanian, “Solid tumors in multiple myeloma,” Ann Intern Med. 1979;90(5):780-2). In some embodiments, the cancer is non-small cell lungcancer. In some embodiments, the cancer is renal cell carcinoma. In someembodiments, the cancer is urothelial bladder cancer. In someembodiments, the cancer is a solid form of a newly diagnosed or arecurrent cancer selected from the group consisting of a blastoma, acarcinoma, a glioma, a leukemia, a lymphoma, a melanoma, a myeloma, anda sarcoma. In some embodiments, the cancer is selected from the groupconsisting of colorectal cancer, diffuse large B-cell lymphoma,follicular lymphoma, glioblastoma, lower grade gliomas, melanoma,multiple myeloma, non-small cell lung cancer, renal cell carcinoma,urothelial bladder cancer, ovarian cystadenocarcinoma, stomachadenocarcinoma and other gastrointestinal malignancies, head and neckadenocarcinoma, pancreatic adenocarcinoma, prostate adenocarcinoma,breast adenocarcinoma, breast cancer and pancreatic cancer.

A “tumor,” as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all precancerous andcancerous cells and tissues. A “solid tumor”, as used herein, is anabnormal mass of tissue that generally does not contain cysts or liquidareas. A solid tumor may be in the brain, colon, breasts, prostate,liver, kidneys, lungs, esophagus, head and neck, ovaries, cervix,stomach, colon, rectum, bladder, uterus, testes, and pancreas, asnon-limiting examples. In some embodiments, the solid tumor regresses orits growth is slowed or arrested after the solid tumor is treated withthe presently disclosed methods. In other embodiments, the solid tumoris malignant.

A “locally administered chemotherapy” is used to connote a compound orcomposition that is locally administered in the treatment of cancer,such as intratumorally and/or within the tumor bed. In some embodiments,local immunotherapy does not compromise the body's ability to mount animmune response. In some embodiments, locally administered chemotherapyallows generation of a robust immune response, whereas systemicchemotherapy compromises the body's ability to generate an effectiveimmune response.

Locally administered chemotherapy useful in methods, compositions, andkits disclosed herein include, but are not limited to, alkylating agentssuch as thiotepa, temozolomide, and cyclophosphamide; alkyl sulfonatessuch as busulfan, improsulfan and piposulfan; aziridines such asbenzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethylenethiophosphaoramide andtrimethylolomelamime; nitrogen mustards such as chlorambucil,chlornaphazine, cholophosphamide, estramustine, ifosfamide,mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard;nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin,authramycin, azaserine, bleomycins, cactinomycin, calicheamicin,carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin,epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins,mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin,puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin,tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such asmethotrexate and 5-fluorouracil (5-FU); folic acid analogues such asdenopterin, methotrexate, pteropterin, trimetrexate; purine analogs suchas fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine,floxuridine, 5-FU; androgens such as calusterone, dromostanolonepropionate, epitiostanol, mepitiostane, testolactone; anti-adrenals suchas aminoglutethimide, mitotane, trilostane; folic acid replenishers suchas folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (Ara-C); taxoids, e.g. paclitaxel and docetaxel;chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; platinumanalogs such as cisplatin and carboplatin; vinblastine; platinum;etoposide; ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT11; topoisomerase inhibitor RFS 2000;difluoromethylornithine; retinoic acid; esperamicins; capecitabine;immune system blockers, e.g. rapamycin; amino acid modifiers, e.g.asparaginase; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Locally administered chemotherapy alsoincludes anti-hormonal agents that act to regulate or inhibit hormoneaction on tumors such as anti-estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston);and anti-androgens such as flutamide, nilutamide, bicalutamide,leuprolide, and goserelin; and pharmaceutically acceptable salts, acidsor derivatives of any of the above.

In some embodiments, the locally administered chemotherapy is atopoisomerase inhibitor. Topoisomerase inhibitors are chemotherapyagents that interfere with the action of a topoisomerase enzyme (e.g.,topoisomerase I or II). Topoisomerase inhibitors include, but are notlimited to, doxorubicin HCl, daunorubicin citrate, mitoxantrone HCl,actinomycin D, etoposide, topotecan HCl, teniposide, and irinotecan, aswell as pharmaceutically acceptable salts, acids, or derivatives of anyof these.

In some embodiments, the locally administered chemotherapy is ananti-metabolite. An anti-metabolite is a chemical with a structure thatis similar to a metabolite required for normal biochemical reactions,yet different enough to interfere with one or more normal functions ofcells, such as cell division. Anti-metabolites include, but are notlimited to, gemcitabine, fluorouracil, capecitabine, methotrexatesodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside,thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine,6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, aswell as pharmaceutically acceptable salts, acids, or derivatives of anyof these.

In certain embodiments, the locally administered chemotherapy is anantimitotic agent, including, but not limited to, agents that bindtubulin. In some embodiments, the agent is a taxane. In certainembodiments, the agent is paclitaxel or docetaxel, or a pharmaceuticallyacceptable salt, acid, or derivative of paclitaxel or docetaxel. Incertain alternative embodiments, the antimitotic agent comprises a vincaalkaloid, such as vincristine, binblastine, vinorelbine, or vindesine,or pharmaceutically acceptable salts, acids, or derivatives thereof.

In some embodiments, the locally administered chemotherapy is formulatedby loading the locally administered chemotherapy into a lipidparticulate drug delivery system, a polymeric drug delivery system, or acatheter.

Examples of suitable lipid particulate drug delivery systems include,for example, solid lipid nanoparticles, nanostructured lipid carriers,lipid drug conjugate-nanoparticles, liposomes, transfersomes, ethosomes,lipospheres, niosomes, cubosomes, virosomes, iscoms, nanoemulsions,cochleates, and phytosomes. In some embodiments, the lipid particulatedrug delivery system is selected from the group consisting of a solidlipid nanoparticle, a nanostructured lipid carrier, a lipid drugconjugate-nanoparticle, a liposome, a transfersome, an ethosome, aliposphere, a niosome, a cubosome, a virosome, an iscom, a nanoemulsion,a cochleate, and a phytosome.

Examples or suitable polymeric drug delivery systems include, forexample, dendrimers, micelles, polymeric microspheres, polymericnanoparticles, and wafers. Polymer microspheres include those fabricatedfrom PLGA, poly(methylidene malonate), PMM, poly(epsilon-caprolactone),chitosan, and the like. In other embodiments, particularly forintracranial applications, convection-enhanced delivery (CED) ofnanoparticles, in which compositions in nanoparticles are infusedcontinuously into the brain tissue via bulk fluid flow using a syringepump, can be used. Examples of drug delivery wafers include, but are notlimited to, the Gliadel® like device, electrospun/rotary jet-spunwafers, biodegradable polymers giving a sustained drug release, the DCBead®, a composite nanofiber mat electrospun from an emulsion containingPLGA, biodegradable electrospun polymeric implants in the form ofmicrofiber discs and sheets, electrospun PLGA fibers, PLA/PLGAelectrospun fibers, fibrous wafers made up of two different kinds ofpolymeric fibers loaded separately with two different drugs, and thelike. In some embodiments, the locally administered chemotherapycomprises a BCNU implantable wafer. In some embodiments, the polymericdrug delivery system is selected from the group consisting of adendrimer, a micelle, a polymeric microsphere, a polymeric nanoparticle,and a wafer. In some embodiments, the delivery by a catheter is selectedfrom the group consisting of microcatheter delivery and convectionenhanced delivery.

As used herein, the term “immunotherapeutic agent” refers to a moleculethat can aid in the treatment of a disease by inducing, enhancing, orsuppressing an immune response. Examples of immunotherapeutic agentsinclude, but are not limited to, immune checkpoint molecules (e.g.,antibodies to immune checkpoint proteins), interleukins (e.g., IL-2,IL-7, IL-12, IL-15), cytokines (e.g., interferons, G-CSF, imiquimod),chemokines (e.g., CCL3, CCL26, CXCL7), vaccines (e.g., peptide vaccines,dendritic cell (DC) vaccines, EGFRvIII vaccines, mesothilin vaccine,G-VAX, listeria vaccines), and adoptive T cell therapy includingchimeric antigen receptor T cells (CAR T cells).

As used herein, the term “immune checkpoint molecule” refers tomolecules that totally or partially reduce, inhibit, interfere with,activate, or modulate one or more checkpoint proteins (i.e., an immunecheckpoint receptor or a ligand for the immune checkpoint receptor). Insome embodiments, the immune checkpoint molecule is an inhibitormolecule.

Examples of immune checkpoint molecules that can be used in thepresently disclosed methods include, but are not limited to, smallorganic molecules (e.g., haptens) or small inorganic molecules;saccharides; oligosaccharides; polysaccharides; a biologicalmacromolecule selected from the group consisting of peptides (e.g.,aptides), proteins, peptide analogs and derivatives; peptidomimetics;nucleic acids selected from the group consisting of miRNAs, siRNAs,shRNAs, antisense nucleic acids, such as antisense RNAs, ribozymes, andaptamers; an extract made from biological materials selected from thegroup consisting of bacteria, plants, fungi, animal cells, and animaltissues; naturally occurring or synthetic compositions; and anycombination thereof. Other examples of immune checkpoint moleculesinclude orthosteric inhibitors, allosteric regulators, interfacialbinders, and molecular analogues of substrates that act as competitiveinhibitors. Specific examples of immune checkpoint molecules includeanti-PD-1, anti-PD-L1, anti-CTLA-4, anti-Lag-3, Anti-CD137, Anti-KIR,anti-Tim3, anti-Ox40, SP4205, a small molecule blocker for theinteraction between Interleukin-2 (IL-2) and the IL-2 receptor andinhibitors of indoleamine-pyrrole 2,3-dioxygenase (IDO), such as thenatural products cabbage extract brassinin, the marine hydroid extractannulin B, and the marine sponge extract exiguamine A, and 1-methyltryptophan (1-MT), a tryptophan mimetic.

As used interchangeably herein, the terms “nucleic acids,”“oligonucleotides,” and “polynucleotides” include RNA, DNA, or RNA/DNAhybrid sequences of more than one nucleotide in either single chain orduplex form. The term “nucleotide” as used herein as an adjective todescribe molecules comprising RNA, DNA, or RNA/DNA hybrid sequences ofany length in single-stranded or duplex form. The term “nucleotide” isalso used herein as a noun to refer to individual nucleotides orvarieties of nucleotides, meaning a molecule, or individual unit in alarger nucleic acid molecule, comprising a purine or pyrimidine, aribose or deoxyribose sugar moiety, and a phosphate group, orphosphodiester linkage in the case of nucleotides within anoligonucleotide or polynucleotide. The term “nucleotide” is also usedherein to encompass “modified nucleotides” which comprise at least oneof the following modifications: (a) an alternative linking group, (b) ananalogous form of purine, (c) an analogous form of pyrimidine, or (d) ananalogous sugar. For examples of analogous linking groups, purine,pyrimidines, and sugars, see for example PCT Patent App. Pub. No. WO95/04064. The polynucleotide sequences of the presently disclosedsubject matter may be prepared by any known method, including synthetic,recombinant, ex vivo generation, or a combination thereof, as well asutilizing any purification methods known in the art.

The term “antisense nucleic acid” refers to an oligonucleotide that hasa nucleotide sequence that interacts through base pairing with aspecific complementary nucleic acid sequence involved in the expressionof the target such that the expression of the gene is reduced.Preferably, the specific nucleic acid sequence involved in theexpression of the gene is a genomic DNA molecule or mRNA molecule thatencodes (a part of) the gene. This genomic DNA molecule can compriseregulatory regions of the gene, or the coding sequence for the maturegene.

The down regulation of gene expression using antisense nucleic acids canbe achieved at the translational or transcriptional level using anexpression-inhibitory agent. Antisense nucleic acids of the inventionare preferably nucleic acid fragments capable of specificallyhybridizing with all or part of a nucleic acid encoding a protein kinaseor the corresponding messenger gene or mRNA. The preparation and use ofantisense nucleic acids, DNA encoding antisense RNAs and the use ofoligo and genetic antisense is known in the art.

Small interfering RNA (siRNA) mediate the post-transcriptional processof gene silencing by double stranded RNA (dsRNA) that is homologous insequence to the silenced RNA. A small hairpin RNA or short hairpin RNA(shRNA) is an artificial RNA molecule with a tight hairpin turn. AmicroRNA (miRNA) is a small non-coding RNA molecule which also functionsin RNA silencing.

Ribozymes are catalytic RNA molecules (RNA enzymes) that have separatecatalytic and substrate binding domains. The substrate binding sequencecombines by nucleotide complementarity and, possibly, non-hydrogen bondinteractions with its mRNA sequence. The catalytic portion cleaves themRNA at a specific site.

As used herein, “expression” refers to the process by which apolynucleotide is transcribed from a DNA template (such as into an mRNAor other RNA transcript) and/or the process by which a transcribed mRNAis subsequently translated into peptides, polypeptides, or proteins. Theterm “polypeptide” or “protein” as used herein refers to a moleculecomprising a string of at least three amino acids linked together bypeptide bonds. The terms “protein” and “polypeptide” may be usedinterchangeably. Proteins may be recombinant or naturally derived.

As used herein, the term “reduce” or “inhibit,” and grammaticalderivations thereof, refers to the ability of an agent to block,partially block, interfere, decrease, reduce or deactivate a biologicalmolecule, pathway or mechanism of action. Thus, one of ordinary skill inthe art would appreciate that the term “inhibit” encompasses a completeand/or partial loss of activity, e.g., a loss in activity by at least10%, in some embodiments, a loss in activity by at least 20%, 30%, 50%,75%, 95%, 98%, and up to and including 100%.

As used herein, the terms “immune checkpoint protein” and “checkpointprotein” are used synonymously to refer to a molecule that transmits aninhibitory signal to an immune cell. In some embodiments, checkpointproteins regulate T-cell activation or function. Numerous checkpointproteins are known, such as CTLA-4 and its ligands CD 80 and CD86; andPD1 with its ligands PDL1 and PDL2. These proteins are responsible forco-stimulatory or inhibitory interactions of T-cell responses. Immunecheckpoint proteins regulate and maintain self-tolerance and theduration and amplitude of physiological immune responses. With respectto an immune checkpoint, the term “activity” includes the ability of animmune checkpoint to modulate an inhibitory signal in an activatedimmune cell, e.g., by engaging an immune checkpoint ligand on an antigenpresenting cell. Modulation of an inhibitory signal in an immune cellresults in modulation of proliferation of, and/or cytokine secretion by,an immune cell. Thus, the term “an immune checkpoint activity” includesthe ability of an immune checkpoint to bind its natural ligand(s), theability to modulate immune cell costimulatory or inhibitory signals, andthe ability to modulate the immune response.

PD1 (Programmed Cell Death Protein 1; e.g. GenBank Accession No.NP_005009.2), also known as CD279 (Cluster of Differentiation 279), is acell surface membrane protein that is expressed mainly on a subset ofactivated T lymphocytes. In humans, it is encoded by the PDCD1 gene(Entrez Gene GeneID: 5133). PD1 is a member of the immunoglobulin genesuperfamily, and has an extracellular region containing immunoglobulinsuperfamily domain, a transmembrane domain, and an intracellular regionincluding an immunoreceptor tyrosine-based inhibitory motif. PD1 israpidly induced on the surface of T-cells in response to anti-CD3. PD1is also induced on the surface of B-cells (in response to anti-IgM) andis expressed on a subset of thymocytes and myeloid cells. Two types ofhuman PD1 ligands have been identified: PDL1 and PDL2. PD1 ligandscomprise a signal sequence, and an IgV domain, an IgC domain, atransmembrane domain, and a short cytoplasmic tail. Both PDL1 (NCBIReference Sequence: NP_001254635.1) and PDL2 (NCBI Reference Sequence:NP_079515.2) are members of the B7 family of polypeptides.

In some embodiments, the immune checkpoint receptor is PD1. In someembodiments, the immune checkpoint molecule comprises an anti-PD1antibody. Examples of anti-PD1 antibodies of use in the presentlydisclosed methods, compositions, and kits include, without limitation,AMP-224, lambrolizumab, nivolumab, and pidilizumab, as shown in Table 1below. In some embodiments, the anti-PD1 antibody is selected from thegroup consisting of AMP-224, lambrolizumab, nivolumab, and pidilizumab.

In some embodiments, the ligand for the immune checkpoint receptor isselected from the group consisting of PDL1 and PDL2. In someembodiments, the immune checkpoint molecule is an anti-PDL1 antibody.Examples of anti-PDL1 antibodies of use in the presently disclosedmethods, compositions, and kits include, without limitation, BMS-936559,MEDI-4736, and MPDL3280A as shown in Table 1 below. In some embodiments,the anti-PDL1 antibody is selected from the group consisting ofBMS-936559, MEDI-4736, and MPDL3280A.

TABLE 1 Exemplary Immune Checkpoint Proteins and Immune CheckpointMolecules State of clinical Biological Antibody or Ig development as ofTarget function fusion protein January 2012 CTLA4 Inhibitory IpilimumabFDA approved for receptor melanoma, Phase II and Phase III trialsongoing for multiple cancers Tremelimumab Previously tested in a PhaseIII trial of patients with melanoma PD1 Inhibitory Nivolumab (BMS- PhaseI/II trials in patients receptor 936558, MDX- with melanoma and renal1106, ONO-4538) and lung cancers fully human Immunoglobulin G4 (IgG4)monoclonal PD-1 antibody Lambrolizumab Phase I trial in multiple(MK3475) cancers humanized monoclonal IgG4 PD-1 antibody Pidilizumab(CT- Phase I trial in multiple 011) cancers humanized monoclonalantibody (mAb) AMP-224 (PDL2- Phase I trial in multiple Ig fusionprotein cancers that blocks PD1 from binding its partners) PDL1 Ligandfor MDX-1105 Phase I trial in multiple PD1 cancers Multiple mAbs Phase Itrials planned for 2012 LAG3 Inhibitory IMP321 (LAG3-Ig Phase III trialin breast receptor fusion protein) cancer Multiple mAbs Preclinicaldevelopment B7-H3 Inhibitory MGA271 Phase I trial in multiple ligandcancers B7-H4 Inhibitory Preclinical development ligand TIM3 InhibitoryPreclinical development receptor CTLA4, cytotoxicT-lymphocyte-associated antigen 4; FDA, US Food and Drug Administration;Ig, immunoglobulin; LAG3, lymphocyte activation gene 3; mAbs, monoclonalantibodies; PD1, programmed cell death protein 1; PDL, PD1 ligand; TIM3,T cell membrane protein 3.

The term “antibody,” also known as an immunoglobulin (Ig), is a largeY-shaped protein produced by B cells that is used by the immune systemto identify and neutralize foreign objects such as bacteria and virusesby recognizing a unique portion (epitope) of the foreign target, calledan antigen. As used herein, the term “antibody” also includes an“antigen-binding portion” of an antibody (or simply “antibody portion”).The term “antigen-binding portion,” as used herein, refers to one ormore fragments of an antibody that retain the ability to specificallybind to an antigen (e.g., PD1). It has been shown that theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “antigen-binding portion” of an antibody include: (i) a Fabfragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the VH and CH1 domains; (iv) a Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody; (v)a dAb fragment, which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, VL and VH, are coded for by separate genes,they can be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent polypeptides (known as singlechain Fv (scFv)). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.Any VH and VL sequences of specific scFv can be linked to humanimmunoglobulin constant region cDNA or genomic sequences, in order togenerate expression vectors encoding complete IgG polypeptides or otherisotypes. VH and V1 can also be used in the generation of Fab, Fv orother fragments of immunoglobulins using either protein chemistry orrecombinant DNA technology. Other forms of single chain antibodies, suchas diabodies are also encompassed. Diabodies are bivalent, bispecificantibodies in which VH and VL domains are expressed on a singlepolypeptide chain, but using a linker that is too short to allow forpairing between the two domains on the same chain, thereby forcing thedomains to pair with complementary domains of another chain and creatingtwo antigen binding sites (e.g., Holliger et al. (1993) Proc. Natl.Acad. Sci. USA 90:6444-6448; Poljak et al. (1994) Structure2:1121-1123).

Still further, an antibody or antigen-binding portion thereof may bepart of larger immunoadhesion polypeptides, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionpolypeptides include use of the streptavidin core region to make atetrameric scFv polypeptide (Kipriyanov et al. (1995) Human Antibodiesand Hybridomas 6:93-101) and use of a cysteine residue, a marker peptideand a C-terminal polyhistidine tag to make bivalent and biotinylatedscFv polypeptides (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058).Antibody portions, such as Fab and F(ab′)₂ fragments, can be preparedfrom whole antibodies using conventional techniques, such as papain orpepsin digestion, respectively, of whole antibodies. Moreover,antibodies, antibody portions and immunoadhesion polypeptides can beobtained using standard recombinant DNA techniques, as described herein.

Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, orsyngeneic; or modified forms thereof (e.g. humanized, chimeric, etc.).Antibodies may also be fully human. Preferably, antibodies of thepresently disclosed subject matter bind specifically or substantiallyspecifically to an immune checkpoint protein or functional variantsthereof. The terms “monoclonal antibodies” and “monoclonal antibodycomposition,” as used herein, refer to a population of antibodypolypeptides that contain only one species of an antigen binding sitecapable of immunoreacting with a particular epitope of an antigen,whereas the term “polyclonal antibodies” and “polyclonal antibodycomposition” refer to a population of antibody polypeptides that containmultiple species of antigen binding sites capable of interacting with aparticular antigen. A monoclonal antibody composition typically displaysa single binding affinity for a particular antigen with which itimmunoreacts.

The term “humanized antibody”, as used herein, is intended to includeantibodies made by a non-human cell having variable and constant regionswhich have been altered to more closely resemble antibodies that wouldbe made by a human cell. For example, by altering the non-human antibodyamino acid sequence to incorporate amino acids found in human germlineimmunoglobulin sequences. The humanized antibodies of the presentlydisclosed subject matter may include amino acid residues not encoded byhuman germline immunoglobulin sequences (e.g., mutations introduced byrandom or site-specific mutagenesis in vitro or by somatic mutation invivo), for example in the CDRs. The term “humanized antibody”, as usedherein, also includes antibodies in which CDR sequences derived from thegermline of another mammalian species, such as a mouse, have beengrafted onto human framework sequences.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds an immune checkpoint protein is substantially free of antibodiesthat specifically bind antigens other than the immune checkpointprotein). Moreover, an isolated antibody may be substantially free ofother cellular material and/or chemicals.

An isolated immune checkpoint protein or functional variant thereof (ora nucleic acid encoding such polypeptides), can be used as an immunogento generate antibodies that bind to the respective immune checkpointprotein or functional variant thereof using standard techniques forpolyclonal and monoclonal antibody preparation. A full-length immunecheckpoint protein can be used, or alternatively, the presentlydisclosed subject matter relates to antigenic peptide fragments of animmune checkpoint protein (e.g., receptor or ligand) or functionalvariants thereof for use as immunogens. An antigenic peptide of animmune checkpoint protein or a functional variant thereof comprises atleast 8 amino acid residues and encompasses an epitope present in therespective full length molecule such that an antibody raised against thepeptide forms a specific immune complex with the respective full lengthmolecule. Preferably, the antigenic peptide comprises at least 10 aminoacid residues, more preferably at least 15 amino acid residues, evenmore preferably at least 20 amino acid residues, and most preferably atleast 30 amino acid residues. Preferred epitopes encompassed by theantigenic peptides are regions of an immune checkpoint protein or afunctional variant thereof that are located on the surface of theprotein, e.g., hydrophilic regions. A standard hydrophobicity analysisof the polypeptide molecule can be performed to identify hydrophilicregions. Highly preferred epitopes encompassed by the antigenic peptidesare the regions of the polypeptide molecule which are in theextracellular domain, and therefore are involved in binding. In oneembodiment, such epitopes can be specific for a given polypeptidemolecule from one species, such as mouse or human (i.e., an antigenicpeptide that spans a region of the polypeptide molecule that is notconserved across species is used as immunogen; such non conservedresidues can be determined using an alignment such as that providedherein).

An immunogen comprising an immune checkpoint protein or a functionalvariant thereof typically is used to prepare antibodies by immunizing asuitable subject, (e.g., rabbit, goat, mouse or other mammal) with theimmunogen. An appropriate immunogenic preparation can contain, forexample, a recombinantly expressed or chemically synthesized molecule orfragment thereof to which the immune response is to be generated. Thepreparation can further include an adjuvant, such as Freund's completeor incomplete adjuvant, or similar immunostimulatory agent. Immunizationof a suitable subject with an immunogenic preparation induces apolyclonal antibody response to the antigenic peptide contained therein.

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with a polypeptide immunogen. The polypeptide antibodytiter in the immunized subject can be monitored over time by standardtechniques, such as with an enzyme linked immunosorbent assay (ELISA)using immobilized polypeptide. If desired, the antibody directed againstthe antigen can be isolated from the mammal (e.g., from the blood) andfurther purified by well known techniques, such as protein Achromatography to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495-497; Brown et al. (1981) J. Immunol. 127:539-46; Brown etal. (1980) J. Biol. Chem. 255:4980-83; Yeh et al. (1976) Proc. Natl.Acad. Sci. 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75),a human B cell hybridoma technique (Kozbor et al. (1983) Immunol. Today4:72), the EBV-hybridoma technique (Cole et al. (1985) MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or triomatechniques. The technology for producing monoclonal antibody hybridomasis well known (see generally Kenneth, R. H. in Monoclonal Antibodies: ANew Dimension In Biological Analyses, Plenum Publishing Corp., New York,N.Y. (1980); Lerner (1981) Yale J. Biol. Med. 54:387-402; Gefter et al.(1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line(typically a myeloma) is fused to lymphocytes (typically splenocytes)from a mammal immunized with an immunogen as described above, and theculture supernatants of the resulting hybridoma cells are screened toidentify a hybridoma producing a monoclonal antibody that binds to thepolypeptide antigen, preferably specifically.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating amonoclonal antibody to an immune checkpoint protein (e.g., Galfre, G. etal. (1977) Nature 266:55052; Kenneth, R. H. in Monoclonal Antibodies: ANew Dimension In Biological Analyses, Plenum Publishing Corp., New York,N.Y. (1980); Lerner (1981) Yale J. Biol. Med. 54:387-402; Gefter et al.(1977) Somatic Cell Genet. 3:231-36). Moreover, the ordinary skilledworker will appreciate that there are many variations of such methodswhich also would be useful. Typically, the immortal cell line (e.g., amyeloma cell line) is derived from the same mammalian species as thelymphocytes. For example, murine hybridomas can be made by fusinglymphocytes from a mouse immunized with an immunogenic preparation ofthe present presently disclosed subject matter with an immortalizedmouse cell line. Preferred immortal cell lines are mouse myeloma celllines that are sensitive to culture medium containing hypoxanthine,aminopterin and thymidine (“HAT medium”). Any of a number of myelomacell lines can be used as a fusion partner according to standardtechniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O—Ag14myeloma lines. These myeloma lines are available from the American TypeCulture Collection (ATCC), Rockville, Md. Typically, HAT-sensitive mousemyeloma cells are fused to mouse splenocytes using polyethylene glycol(“PEG”). Hybridoma cells resulting from the fusion are then selectedusing HAT medium, which kills unfused and unproductively fused myelomacells (unfused splenocytes die after several days because they are nottransformed). Hybridoma cells producing a monoclonal antibody of thepresently disclosed subject matter are detected by screening thehybridoma culture supernatants for antibodies that bind a givenpolypeptide, e.g., using a standard ELISA assay.

As an alternative to preparing monoclonal antibody-secreting hybridomas,a monoclonal specific for one of the above described polypeptidesantibody can be identified and isolated by screening a recombinantcombinatorial immunoglobulin library (e.g., an antibody phage displaylibrary) with the appropriate polypeptide to thereby isolateimmunoglobulin library members that bind the polypeptide. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit,Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening an antibodydisplay library can be found in, for example, U.S. Pat. No. 5,223,409;PCT Patent App. Pub. No. WO 92/18619; PCT Patent App. Pub. No. WO91/17271; PCT Patent App. Pub. No. 92/20791; PCT Patent App. Pub. No. WO92/15679; PCT Patent App. Pub. No. WO 93/01288; PCT Patent App. Pub. No.WO 92/01047; PCT Patent App. Pub. No. WO 92/09690; PCT Patent App. Pub.No. WO 90/02809; Fuchs et al. (1991) Biotechnology (NY) 9:1369-1372; Hayet al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989)Science 246:1275-1281; Griffiths et al. (1993) EMBO J 12:725-734;Hawkins et al. (1992) J Mol. Biol. 226:889-896; Clarkson et al. (1991)Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA89:3576-3580; Garrard et al. (1991) Biotechnology (NY) 9:1373-1377;Hoogenboom et al. (1991) Nucleic Acids Res. 19:4133-4137; Barbas et al.(1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and McCafferty et al.(1990) Nature 348:552-554.

Additionally, recombinant immunotherapeutic agents, such as immunecheckpoint molecules, such as chimeric and humanized monoclonalantibodies, comprising both human and non-human portions, which can bemade using standard recombinant DNA techniques, are within the scope ofthe presently disclosed subject matter. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in PCT Patent App.Pub. No. PCT/US86/02269; European Patent App. No. 184,187; EuropeanPatent App. No. 171,496; European Patent App. No. 173,494; PCTApplication WO 86/01533; U.S. Pat. No. 4,816,567; European Patent App.No. 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al.(1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci.84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; and Shaw et al. (1988) J Natl. Cancer Inst.80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al.(1986) Biotechniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986)Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

In addition, humanized antibodies can be made according to standardprotocols such as those disclosed in U.S. Pat. No. 5,565,332. In anotherembodiment, antibody chains or specific binding pair members can beproduced by recombination between vectors comprising nucleic acidmolecules encoding a fusion of a polypeptide chain of a specific bindingpair member and a component of a replicable generic display package andvectors containing nucleic acid molecules encoding a second polypeptidechain of a single binding pair member using techniques known in the art,e.g., as described in U.S. Pat. Nos. 5,565,332, 5,871,907, or 5,733,743.The use of intracellular antibodies to inhibit protein function in acell is also known in the art (e.g., Carlson (1988) Mol. Cell. Biol.8:2638-2646; Biocca et al. (1990) EMBO J. 9:101-108; Werge et al. (1990)FEBS Lett 274:193-198; Carlson (1993) Proc. Natl. Acad. Sci. USA90:7427-7428; Marasco et al. (1993) Proc. Natl. Acad. Sci. USA90:7889-7893; Biocca et al. (1994) Biotechnology (NY) 12:396-399; Chenet al. (1994) Hum. Gene Ther. 5:595-601; Duan et al. (1994) Proc. Natl.Acad. Sci. USA 91:5075-5079; Chen et al. (1994) Proc. Natl. Acad. Sci.USA 91:5932-5936; Beerli et al. (1994) J. Biol. Chem. 269:23931-23936;Beerli et al. (1994) Biochem. Biophys. Res. Commun. 204:666-672;Mhashilkar et al. (1995) EMBO J. 14:1542-1551; Richardson et al. (1995)Proc. Natl. Acad. Sci. USA 92:3137-3141; PCT Publication No. WO94/02610; and PCT Publication No. WO 95/03832).

Additionally, fully human antibodies could be made against an immunecheckpoint protein or a functional variant thereof. Fully humanantibodies can be made in mice that are transgenic for humanimmunoglobulin genes, e.g. according to Hogan, et al., “Manipulating theMouse Embryo: A Laboratory Manual,” Cold Spring Harbor Laboratory.Briefly, transgenic mice are immunized with a purified immune checkpointprotein or a functional variant thereof. Spleen cells are harvested andfused to myeloma cells to produce hybridomas. Hybridomas are selectedbased on their ability to produce antibodies which bind to an immunecheckpoint protein or a functional variant thereof. Fully humanantibodies would reduce the immunogenicity of such antibodies in ahuman.

In one embodiment, an antibody for use in the instant presentlydisclosed subject matter is a bispecific antibody. A bispecific antibodyhas binding sites for two different antigens within a single antibodypolypeptide. Antigen binding may be simultaneous or sequential. Triomasand hybrid hybridomas are two examples of cell lines that can secretebispecific antibodies. Examples of bispecific antibodies produced by ahybrid hybridoma or a trioma are disclosed in U.S. Pat. No. 4,474,893.Bispecific antibodies have been constructed by chemical means (Staerz etal. (1985) Nature 314:628, and Perez et al. (1985) Nature 316:354) andhybridoma technology (Staerz and Bevan (1986) Proc. Natl. Acad. Sci.USA, 83:1453, and Staerz and Bevan (1986) Immunol. Today 7:241).Bispecific antibodies are also described in U.S. Pat. No. 5,959,084.Fragments of bispecific antibodies are described in U.S. Pat. No.5,798,229.

Bispecific agents can also be generated by making heterohybridomas byfusing hybridomas or other cells making different antibodies, followedby identification of clones producing and co-assembling both antibodies.They can also be generated by chemical or genetic conjugation ofcomplete immunoglobulin chains or portions thereof such as Fab and Fvsequences. The antibody component can bind to an immune checkpointprotein or a functional variant thereof. In one embodiment, thebispecific antibody could specifically bind to both an immune checkpointreceptor ligand or a functional variant thereof and an immune checkpointreceptor or a functional variant thereof.

Yet another aspect of the presently disclosed subject matter pertains toantibodies that are obtainable by a process comprising, immunizing ananimal with an immunogenic immune checkpoint protein or a functionalvariant thereof, or an immunogenic portion thereof unique to the immunecheckpoint protein, and then isolating from the animal antibodies thatspecifically bind to the polypeptide.

In some embodiments, other immunoregulatory entities can be combinedwith antibodies against an immune checkpoint protein. Suchimmunoregulatory entities may include, for example, immunostimulatorycytokines such as GM-CSF, Interleukin-12 (IL-12), and IL-15.

“Functional variants” of immune checkpoints proteins include functionalfragments, functional mutant proteins, and/or functional fusionproteins. A functional variant of a selected polypeptide refers to anisolated and/or recombinant protein or polypeptide which has at leastone property, activity and/or functional characteristic of the selectedpolypeptide (e.g., PD1). As used herein, the term “activity,” when usedwith respect to a polypeptide, e.g., PD1, includes activities which areinherent in the structure of the wild-type protein.

Generally, fragments or portions of an immune checkpoint proteinencompassed by the presently disclosed subject matter include thosehaving a deletion (i.e. one or more deletions) of an amino acid (i.e.,one or more amino acids) relative to the wild-type immune checkpointprotein (such as N-terminal, C-terminal or internal deletions).Fragments or portions in which only contiguous amino acids have beendeleted or in which non-contiguous amino acids have been deletedrelative to a wild-type immune checkpoint protein are also envisioned.Generally, mutants or derivatives of immune checkpoint proteinsencompassed by the present presently disclosed subject matter includenatural or artificial variants differing by the addition, deletionand/or substitution of one or more contiguous or non-contiguous aminoacid residues, or modified polypeptides in which one or more residues ismodified, and mutants comprising one or more modified residues.Preferred mutants are natural or artificial variants of immunecheckpoint proteins differing by the addition, deletion and/orsubstitution of one or more contiguous or non-contiguous amino acidresidues.

Generally, a functional variant of an immune checkpoint protein has anamino acid sequence which is at least about 80% identical, at leastabout 81% identical, at least about 82% identical, at least about 83%identical, at least about 84% identical, at least about 85% identical,at least about 86% identical, at least about 87% identical, at leastabout 88% identical, at least about 89% identical, at least about 90%identical, at least about 91% identical, at least about 92% identical,at least about 93% identical, at least about 94% identical, at leastabout 95% identical, at least about 96% identical, at least about 97%identical, at least about 98% identical, or at least about 99% identicalto the wild-type amino acid sequence for an immune checkpoint proteinover the length of the variant.

“Sequence identity” or “identity” in the context of proteins orpolypeptides refers to the amino acid residues in two amino acidsequences that are the same when aligned for maximum correspondence overa specified comparison window.

Thus, “percentage of sequence identity” refers to the value determinedby comparing two optimally aligned sequences over a comparison window,wherein the portion of the amino acid sequence in the comparison windowmay comprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the window of comparison and multiplying the results by100 to yield the percentage of sequence identity. Useful examples ofpercent sequence identities include, but are not limited to, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentagefrom 50% to 100%. These identities can be determined using any of theprograms described herein.

Sequence alignments and percent identity or similarity calculations maybe determined using a variety of comparison methods designed to detecthomologous sequences including, but not limited to, the MegAlign™program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.,Madison, Wis.). Within the context of this application it will beunderstood that where sequence analysis software is used for analysis,that the results of the analysis will be based on the “default values”of the program referenced, unless otherwise specified. As used herein“default values” will mean any set of values or parameters thatoriginally load with the software when first initialized. The “Clustal Vmethod of alignment” corresponds to the alignment method labeled ClustalV (described by Higgins and Sharp (1989) CABIOS 5:151-153; Higgins etal. (1992) Comput. Appl. Biosci. 8:189-191) and found in the MegAlign™program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.,Madison, Wis.).

It is well understood by one skilled in the art that many levels ofsequence identity are useful in identifying proteins or polypeptides(e.g., from other species) wherein the proteins or polypeptides have thesame or similar function or activity. Useful examples of percentidentities include, but are not limited to, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, or 95%, or any integer percentage from 50% to 100%.Indeed, any integer amino acid identity from 50% to 100% may be usefulin describing the present presently disclosed subject matter, such as51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99%.

The terms “subject” and “patient” are used interchangeably herein. Thesubject treated by the presently disclosed methods in their manyembodiments is desirably a human subject, although it is to beunderstood that the methods described herein are effective with respectto all vertebrate species, which are intended to be included in the term“subject.” Accordingly, a “subject” can include a human subject formedical purposes, such as for the treatment of an existing condition ordisease or the prophylactic treatment for preventing the onset of acondition or disease, or an animal subject for medical, veterinarypurposes, or developmental purposes. Suitable animal subjects includemammals including, but not limited to, primates, e.g., humans, monkeys,apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines,e.g., sheep and the like; caprines, e.g., goats and the like; porcines,e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras,and the like; felines, including wild and domestic cats; canines,including dogs; lagomorphs, including rabbits, hares, and the like; androdents, including mice, rats, and the like. An animal may be atransgenic animal. In some embodiments, the subject is a humanincluding, but not limited to, fetal, neonatal, infant, juvenile, andadult subjects. Further, a “subject” can include a patient afflictedwith or suspected of being afflicted with a condition or disease.

Aspects of the presently disclosed subject matter relate toimmunotherapeutic, non-immunosuppressive compositions comprising alocally administered chemotherapy and/or an immunotherapeutic agent,such as an immune checkpoint molecule, formulated for localadministration (e.g., intratumoral). In some embodiments, the presentlydisclosed subject matter provides an immunotherapeutic,non-immunosuppressive composition comprising: (a) a locally administeredchemotherapy; and (b) an immunotherapeutic agent.

The presently disclosed subject matter also contemplates the use of suchimmunotherapeutic, non-immunosuppressive compositions for the treatmentof a cancer. In some embodiments, the presently disclosed methodscomprise the use of the presently disclosed immunotherapeutic,non-immunosuppressive compositions for the manufacture of a medicamentfor the treatment of a cancer. Generally, the presently disclosedcompositions (e.g., comprising an immunotherapeutic agent) can beadministered to a subject for therapy by any suitable route ofadministration, including orally, nasally, transmucosally, ocularly,rectally, intravaginally, parenterally, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intra-articular, intra-sternal,intra-synovial, intra-hepatic, intralesional, intracranial,intraperitoneal, intranasal, or intraocular injections,intracisternally, topically, as by powders, ointments or drops(including eyedrops), including buccally and sublingually,transdermally, through an inhalation spray, or other modes of deliveryknown in the art. However, in some particular embodiments, the presentlydisclosed compositions are administered locally, such as intratumorally,so that the compositions are directly administered into a solid tumor(or injected or implanted into a microenvironment in which the solidtumor resides). In some embodiments, intratumoral administrationcomprises injection into a solid tumor of the patient or injection orimplantation into a microenvironment in which the solid tumor resides orresided. The means of administration into a solid tumor include aneedle, needle-less injection device, or any other means by which theimmunotherapeutic agent and locally administered chemotherapy can beadministered locally. It should be appreciated that all or a portion ofthe solid tumor may be surgically removed prior to locally administeredchemotherapy and/or immunotherapeutic agent. In some embodiments, themethods further comprise surgically removing all or a portion of thesolid tumor prior to locally administered chemotherapy. In someembodiments, the immunotherapeutic agent and the locally administeredchemotherapy are delivered to the tumor site using drug delivery wafers,either separately or together.

The phrases “systemic administration”, “administered systemically”,“peripheral administration” and “administered peripherally” as usedherein mean the administration of compositions comprising at least oneimmunotherapeutic agent combined with at least one locally administeredchemotherapy, such that they enter the patient's system and, thus, aresubject to metabolism and other like processes, for example,subcutaneous administration.

The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intarterial, intrathecal,intracapsular, intraorbital, intraocular, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion.

The phrases “local administration” and “administered locally” as usedherein mean the administration of compositions comprising at least oneimmunotherapeutic agent combined with at least one locally administeredchemotherapy, such that they enter into a patient's solid tumor and thearea surrounding the tumor (i.e., tumor microenvironment), withoutentering into the rest of the patient's system. In some embodiments, theadministration of compositions allows the immunotherapeutic agent andlocally administered chemotherapy to be distributed over a largerregional area, e.g. through large volumes of brain tissue.

The presently disclosed pharmaceutical compositions can be manufacturedin a manner known in the art, e.g. by means of conventional mixing,dissolving, granulating, dragee-making, levitating, emulsifying,encapsulating, entrapping or lyophilizing processes.

In some embodiments, the presently disclosed pharmaceutical compositionscan be administered by rechargeable or biodegradable devices. Forexample, a variety of slow-release polymeric devices have been developedand tested in vivo for the controlled delivery of drugs, includingproteinacious biopharmaceuticals. Suitable examples of sustained releasepreparations include semipermeable polymer matrices in the form ofshaped articles, e.g., films or microcapsules. Sustained releasematrices include polyesters, hydrogels, polylactides (U.S. Pat. No.3,773,919; EP 58,481), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., Biopolymers 22:547, 1983), poly(2-hydroxyethyl-methacrylate) (Langer et al. (1981) Biomed. Mater. Res.15:167; Langer (1982), Chem. Tech. 12:98), ethylene vinyl acetate(Langer et al. (1981) J Biomed. Mater. Res. 15:167), orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988A). Sustained releasecompositions also include liposomally entrapped compositions comprisingat least one immunotherapeutic agent combined with at least one locallyadministered chemotherapy which can be prepared by methods known in theart (Epstein et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:3688; Hwanget al. (1980) Proc. Natl. Acad. Sci. U.S.A. 77:4030; U.S. Pat. Nos.4,485,045 and 4,544,545; and EP 102,324A). Ordinarily, the liposomes areof the small (about 200-800 angstroms) unilamelar type in which thelipid content is greater than about 30 mol % cholesterol, the selectedproportion being adjusted for the optimal therapy. Such materials cancomprise an implant, for example, for sustained release of the presentlydisclosed compositions, which, in some embodiments, can be implanted ata particular, pre-determined target site, such as at a solid tumor, orat a site at which a solid tumor has been surgically removed.

In another embodiment, the presently disclosed pharmaceuticalcompositions may comprise PEGylated therapeutics (e.g., PEGylatedantibodies). PEGylation is a well-established and validated approach forthe modification of a range of antibodies, proteins, and peptides andinvolves the attachment of polyethylene glycol (PEG) at specific sitesof the antibodies, proteins, and peptides (Chapman (2002) Adv. DrugDeliv. Rev. 54:531-545). Some effects of PEGylation include: (a)markedly improved circulating half-lives in vivo due to either evasionof renal clearance as a result of the polymer increasing the apparentsize of the molecule to above the glomerular filtration limit, and/orthrough evasion of cellular clearance mechanisms; (b) improvedpharmacokinetics; (c) improved solubility—PEG has been found to besoluble in many different solvents, ranging from water to many organicsolvents such as toluene, methylene chloride, ethanol and acetone; (d)PEGylated antibody fragments can be concentrated to 200 mg/ml, and theability to do so opens up formulation and dosing options such assubcutaneous administration of a high protein dose; this is in contrastto many other therapeutic antibodies which are typically administeredintravenously; (e) enhanced proteolytic resistance of the conjugatedprotein (Cunningham-Rundles et. al. (1992) J. Immunol. Meth.152:177-190); (0 improved bioavailability via reduced losses atsubcutaneous injection sites; (g) reduced toxicity has been observed;for agents where toxicity is related to peak plasma level, a flatterpharmacokinetic profile achieved by sub-cutaneous administration ofPEGylated protein is advantageous; proteins that elicit an immuneresponse which has toxicity consequences may also benefit as a result ofPEGylation; and (h) improved thermal and mechanical stability of thePEGylated molecule.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of compositions comprising at least oneimmunotherapeutic agent combined with at least one locally administeredchemotherapy. For injection, the presently disclosed pharmaceuticalcompositions can be formulated in aqueous solutions, for example, insome embodiments, in physiologically compatible buffers, such as Hank'ssolution, Ringer's solution, or physiologically buffered saline. Aqueousinjection suspensions can contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of compositions include fatty oils,such as sesame oil, or synthetic fatty acid esters, such as ethyl oleateor triglycerides, or liposomes. Optionally, the suspension also cancontain suitable stabilizers or agents that increase the solubility ofthe compositions comprising at least one immunotherapeutic agentcombined with at least one locally administered chemotherapy to allowfor the preparation of highly concentrated solutions.

For nasal or transmucosal administration generally, penetrantsappropriate to the particular barrier to be permeated are used in theformulation. Such penetrants are generally known in the art.

For inhalation delivery, the agents of the disclosure also can beformulated by methods known to those of skill in the art, and mayinclude, for example, but not limited to, examples of solubilizing,diluting, or dispersing substances such as, saline, preservatives, suchas benzyl alcohol, absorption promoters, and fluorocarbons.

Additional ingredients can be added to compositions for topicaladministration, as long as such ingredients are pharmaceuticallyacceptable and not deleterious to the epithelial cells or theirfunction. Further, such additional ingredients should not adverselyaffect the epithelial penetration efficiency of the composition, andshould not cause deterioration in the stability of the composition. Forexample, fragrances, opacifiers, antioxidants, gelling agents,stabilizers, surfactants, emollients, coloring agents, preservatives,buffering agents, and the like can be present. The pH of the presentlydisclosed topical composition can be adjusted to a physiologicallyacceptable range of from about 6.0 to about 9.0 by adding bufferingagents thereto such that the composition is physiologically compatiblewith a subject's skin.

Regardless of the route of administration selected, the presentlydisclosed compositions are formulated into pharmaceutically acceptabledosage forms such as described herein or by other conventional methodsknown to those of skill in the art.

In general, the “effective amount” or “therapeutically effective amount”of an active agent or drug delivery device refers to the amountnecessary to elicit the desired biological response. As will beappreciated by those of ordinary skill in this art, the effective amountof an agent or device may vary depending on such factors as the desiredbiological endpoint, the agent to be delivered, the composition of theencapsulating matrix, the target tissue, and the like.

The term “combination” is used in its broadest sense and means that asubject is administered at least two agents, more particularly at leastone immunotherapeutic agent combined with at least one locallyadministered chemotherapy. More particularly, the term “in combination”refers to the concomitant administration of two (or more) active agentsfor the treatment of a, e.g., single disease state. As used herein, theactive agents may be combined and administered in a single dosage form,may be administered as separate dosage forms at the same time, or may beadministered as separate dosage forms that are administered alternatelyor sequentially on the same or separate days. In one embodiment of thepresently disclosed subject matter, the active agents are combined andadministered in a single dosage form. In another embodiment, the activeagents are administered in separate dosage forms (e.g., wherein it isdesirable to vary the amount of one but not the other). The singledosage form may include additional active agents for the treatment ofthe disease state. In a further embodiment, at least oneimmunotherapeutic agent is administered before at least one locallyadministered chemotherapy. In a still further embodiment, at least onelocally administered chemotherapy is administered before at least oneimmunotherapeutic agent.

Further, the presently disclosed compositions can be administered aloneor in combination with adjuvants that enhance stability of the agents,facilitate administration of pharmaceutical compositions containing themin certain embodiments, provide increased dissolution or dispersion,increase activity, provide adjuvant therapy, and the like, includingother active ingredients. Advantageously, such combination therapiesutilize lower dosages of the conventional therapeutics, thus avoidingpossible toxicity and adverse side effects incurred when those agentsare used as monotherapies.

The timing of administration of at least one immunotherapeutic agentcombined with at least one locally administered chemotherapy can bevaried so long as the beneficial effects of the combination of theseagents are achieved. Accordingly, the phrase “in combination with”refers to the administration of at least one immunotherapeutic agentcombined with at least one locally administered chemotherapy and,optionally, additional agents either simultaneously, sequentially, or acombination thereof. Therefore, a subject administered a combination ofat least one immunotherapeutic agent in combination with at least onelocally administered chemotherapy and, optionally, additional agents canreceive at least one immunotherapeutic agent combined with at least onelocally administered chemotherapy and, optionally, additional agents atthe same time (i.e., simultaneously) or at different times (i.e.,sequentially, in either order, on the same day or on different days), solong as the effect of the combination of all agents is achieved in thesubject.

When administered sequentially, the agents can be administered within 1,5, 10, 30, 60, 120, 180, 240 minutes or longer of one another. In otherembodiments, agents administered sequentially, can be administeredwithin 1, 2, 3, 4, 5, 10, 15, 20 or more days of one another. Where theagents are administered simultaneously, they can be administered to thesubject as separate pharmaceutical compositions, each comprising eitherat least one immunotherapeutic agent in combination with at least onelocally administered chemotherapy and, optionally, additional agents, orthey can be administered to a subject as a single pharmaceuticalcomposition comprising all agents.

When administered in combination, the effective concentration of each ofthe agents to elicit a particular biological response may be less thanthe effective concentration of each agent when administered alone,thereby allowing a reduction in the dose of one or more of the agentsrelative to the dose that would be needed if the agent was administeredas a single agent. The effects of multiple agents may, but need not be,additive or synergistic. The agents may be administered multiple times.

In some embodiments, when administered in combination, the two or moreagents can have a synergistic effect. As used herein, the terms“synergy,” “synergistic,” “synergistically” and derivations thereof,such as in a “synergistic effect” or a “synergistic combination” or a“synergistic composition” refer to circumstances under which thebiological activity of a combination of an agent and at least oneadditional therapeutic agent is greater than the sum of the biologicalactivities of the respective agents when administered individually.

Synergy can be expressed in terms of a “Synergy Index (SI),” whichgenerally can be determined by the method described by F. C. Kull et al.Applied Microbiology 9, 538 (1961), from the ratio determined by:

Q _(a) Q _(A) +Q _(b) Q _(B)=Synergy Index(SI)

wherein:

Q_(A) is the concentration of a component A, acting alone, whichproduced an end point in relation to component A;

Q_(a) is the concentration of component A, in a mixture, which producedan end point;

Q_(B) is the concentration of a component B, acting alone, whichproduced an end point in relation to component B; and

Q_(b) is the concentration of component B, in a mixture, which producedan end point.

Generally, when the sum of Q_(a)/Q_(A) and Q_(b)/Q_(B) is greater thanone, antagonism is indicated. When the sum is equal to one, additivityis indicated. When the sum is less than one, synergism is demonstrated.The lower the SI, the greater the synergy shown by that particularmixture. Thus, a “synergistic combination” has an activity higher thatwhat can be expected based on the observed activities of the individualcomponents when used alone. Further, a “synergistically effectiveamount” of a component refers to the amount of the component necessaryto elicit a synergistic effect in, for example, another therapeuticagent present in the composition.

In another aspect, the presently disclosed subject matter provides apharmaceutical composition including at least one immunotherapeuticagent combined with at least one locally administered chemotherapy,optionally, additional agents, alone or in combination with one or moreadditional therapeutic agents in admixture with a pharmaceuticallyacceptable excipient.

More particularly, the presently disclosed subject matter provides apharmaceutical composition comprising at least one immunotherapeuticagent combined with at least one locally administered chemotherapy and,optionally, additional agents and a pharmaceutically acceptable carrier.

In therapeutic and/or diagnostic applications, the compounds of thedisclosure can be formulated for a variety of modes of administration,including systemic and topical or localized administration. Techniquesand formulations generally may be found in Remington: The Science andPractice of Pharmacy (20^(th) ed.) Lippincott, Williams and Wilkins(2000).

Use of pharmaceutically acceptable inert carriers to formulate thecompounds herein disclosed for the practice of the disclosure intodosages suitable for systemic administration is within the scope of thedisclosure. With proper choice of carrier and suitable manufacturingpractice, the compositions of the present disclosure, in particular,those formulated as solutions, may be administered parenterally, such asby intravenous injection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe disclosure to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya subject (e.g., patient) to be treated.

For nasal or inhalation delivery, the agents of the disclosure also maybe formulated by methods known to those of skill in the art, and mayinclude, for example, but not limited to, examples of solubilizing,diluting, or dispersing substances, such as saline; preservatives, suchas benzyl alcohol; absorption promoters; and fluorocarbons.

Pharmaceutical compositions suitable for use in the present disclosureinclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.Generally, the compounds according to the disclosure are effective overa wide dosage range. For example, in the treatment of adult humans,dosages from 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg perday, and from 5 to 40 mg per day are examples of dosages that may beused. A non-limiting dosage is 10 to 30 mg per day. The exact dosagewill depend upon the route of administration, the form in which thecompound is administered, the subject to be treated, the body weight ofthe subject to be treated, and the preference and experience of theattending physician.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

The term “instructing” a patient as used herein means providingdirections for applicable therapy, medication, treatment, treatmentregimens, and the like, by any means, but preferably in writing.Instructing can be in the form of prescribing a course of treatment, orcan be in the form of package inserts or other written promotionalmaterial.

The term “promoting” as used herein means offering, advertising,selling, or describing a particular drug, combination of drugs, ortreatment modality, by any means, including writing, such as in the formof package inserts. Promoting herein refers to promotion of thecombination of an immunotherapeutic agent, such as an immune checkpointmolecule, and a chemotherapeutic agent formulated for localadministration (e.g., intratumoral) for an indication, such as thetreatment of cancer (e.g., brain cancer, e.g., glioblastoma), where suchpromoting is authorized by the Food and Drug Administration (FDA) ashaving been demonstrated to be associated with statistically significanttherapeutic efficacy and acceptable safety in a population of subjects.In some embodiments, the presently disclosed subject matter provides amethod of promoting a combination treatment for the treatment of apatient with a cancer, wherein the combination treatment comprises: (a)a locally administered chemotherapy; and (b) an immunotherapeutic agent.In some embodiments, promoting is not authorized by the Food and DrugAdministration (FDA) (or other health regulatory agency, such as theEuropean Medicines Agency (EMA), and promoting is for an off-label use.In some embodiments, the package insert provides instructions to receivecancer treatment with a locally administered chemotherapy in combinationwith an immunotherapeutic agent, such as an immune checkpoint molecule.In some embodiments, the package insert provides instructions to receivecancer treatment with a locally administered chemotherapy in combinationwith an immunotherapeutic agent. In some embodiments, the presentlydisclosed subject matter provides a method of instructing a patient witha cancer by providing instructions to receive a combination treatmentcomprising: (a) a locally administered chemotherapy; and (b) animmunotherapeutic agent, to extend survival of the patient. In someembodiments, the promotion is by a package insert accompanying aformulation comprising the locally administered chemotherapy and theimmunotherapeutic agent. In some embodiments, the promotion is bywritten communication to a physician or health care provider. In someembodiments, the promotion is by oral communication to a physician orhealth care provider. In some embodiments, the promotion is by a packageinsert, wherein the package insert provides instructions to receivecancer treatment with a locally administered chemotherapy in combinationwith an immunotherapeutic agent.

II. Kits for Treating Cancer

The presently disclosed subject matter also relates to kits forpracticing the methods of the presently disclosed subject matter. Ingeneral, a presently disclosed kit contains some or all of thecomponents, reagents, supplies, and the like to practice a methodaccording to the presently disclosed subject matter. In someembodiments, the term “kit” refers to any intended any article ofmanufacture (e.g., a package or a container) comprising at least oneimmunotherapeutic agent, such as an immune checkpoint molecule, at leastone locally administered chemotherapy formulated for localadministration (e.g., intratumoral), and a set of particularinstructions for practicing the methods of the presently disclosedsubject matter. The kit can be packaged in a divided or undividedcontainer, such as a carton, bottle, ampule, tube, etc. The presentlydisclosed compositions can be packaged in dried, lyophilized, or liquidform. Additional components provided can include vehicles forreconstitution of dried components. Preferably all such vehicles aresterile and apyrogenic so that they are suitable for injection into asubject without causing adverse reactions.

In some embodiments, the presently disclosed subject matter provides akit comprising: (a) a locally administered chemotherapy; (b) animmunotherapeutic agent; and (c) a package insert or label withdirections to treat a patient with a cancer by administering acombination treatment comprising the locally administered chemotherapyand the immunotherapeutic agent. Those skilled in the art willappreciate that a kit can be assembled for the treatment of any canceror solid tumor described herein.

Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this presently described subject matter belongs.

Following long-standing patent law convention, the terms “a,” “an,” and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a subject” includes aplurality of subjects, unless the context clearly is to the contrary(e.g., a plurality of subjects), and so forth.

Throughout this specification and the claims, the terms “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise. Likewise, the term “include” andits grammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing amounts, sizes, dimensions,proportions, shapes, formulations, parameters, percentages, parameters,quantities, characteristics, and other numerical values used in thespecification and claims, are to be understood as being modified in allinstances by the term “about” even though the term “about” may notexpressly appear with the value, amount or range. Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thefollowing specification and attached claims are not and need not beexact, but may be approximate and/or larger or smaller as desired,reflecting tolerances, conversion factors, rounding off, measurementerror and the like, and other factors known to those of skill in the artdepending on the desired properties sought to be obtained by thepresently disclosed subject matter. For example, the term “about,” whenreferring to a value can be meant to encompass variations of, in someembodiments, ±100% in some embodiments ±50%, in some embodiments ±20%,in some embodiments ±10%, in some embodiments ±5%, in some embodiments±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from thespecified amount, as such variations are appropriate to perform thedisclosed methods or employ the disclosed compositions.

Further, the term “about” when used in connection with one or morenumbers or numerical ranges, should be understood to refer to all suchnumbers, including all numbers in a range and modifies that range byextending the boundaries above and below the numerical values set forth.The recitation of numerical ranges by endpoints includes all numbers,e.g., whole integers, including fractions thereof, subsumed within thatrange (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5,as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like)and any range within that range.

EXAMPLES

The following Examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. In light of the presentdisclosure and the general level of skill in the art, those of skill canappreciate that the following Examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter. The synthetic descriptions and specific examples thatfollow are only intended for the purposes of illustration, and are notto be construed as limiting in any manner to make compounds of thedisclosure by other methods.

Example 1 Materials and Methods

Cells: GL-261 luciferase positive cells (GL-261 LUC) were purchased fromCaliper Life Sciences (Hopkinton, Mass.). Cells were grown in Dulbecco'sModified Eagle Medium (DMEM, Life Technologies, Grand Island, N.Y.) with10% Fetal Bovine Serum (FBS, Gemini Bio-Products, West Sacramento,Calif.) plus 1% Penicillin/Streptomycin (Life Technologies, GrandIsland, N.Y.) and 100 ug/mL of G418 (Invitrogen, San Diego, Calif.) inan incubator maintained at 37° C. with 5% CO₂. GL-261 ova luciferasepositive cells (G1-261 ova-luc) were kindly donated by Dr. Ollin(University of Minnesota) (Ohlfest et al., 2013). Cells were grown inDMEM, with 10% FBS plus 1% Pen/Strep plus 500 ug/mL of G418.

Tumor model: Female C57BL/6J mice (The Jackson Laboratory, Bar Harbor,Me.), 6 to 8 weeks old, were implanted (Day 0) with GL-261 LUC cells toestablish intracranial gliomas, as previously described (Zeng et al.,2013). Briefly, mice were anesthetized with ketamine/xylazine (100 mg/kgketamine, 10 mg/kg xylazine) and a small midline incision was made toexpose the skull. A burr hole was then drilled directly over thestriatum and 130,000 GL-261 LUC cells were implanted at a depth of 3 mmfrom the cortical surface. The tumor take rate was 100%. Day 7 postimplantation, mice were imaged to assess the progress of tumor growthusing an IVIS platform (In Vivo Imaging System, Caliper Life Sciences,Hopkinton, Mass.). Mice were then stratified into experimental treatmentgroups based on luminescence. Each treatment group had 5 to 15 mice inthe survival experiments. The treatment groups were as follows: control(3% ethanol in PBS administered intra-peritoneally), empty polymer (EP),anti-PD1, LC, i.p. BCNU (3% ethanol in PBS), i.p. BCNU+anti-PD1,LC+anti-PD1. All experiments were repeated at least in triplicate unlessotherwise stated. The experimental schedules are explained in FIG. 1.

Adoptive transfer experiments: For the adoptive transfer experiments, 5week old female B6.SJL-Ptprca Pepcb/BoyJ mice expressing the congenicmarker CD45.1 were used as the recipient mice. Inbred C57BL6/J (B6),B6.129S7-RagltmlMom/J (RAG^(−/−)), B6.SJL-Ptprcb Pepcb/BoyJ (CD45.2) andC57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-I) mice were purchased from TheJackson Laboratory. 6-8 weeks old female RAG−/− OT-I CD45.2 transgenicmice were used as the donor mice.

RAG−/− OT-I CD45.2 mice were lethally anesthetized and their spleenswere harvested and passed through a 40-μM nylon mesh. Red blood celllysis buffer was consecutively applied to remove red blood cells. Cellswere counted, checked for their viability, and washed twice with PBS.Two million cells were transferred in 100 uL of PBS for every recipientmouse via retro-orbital injection.

Mice were implanted with 300,000 G1-261-ova-luc cells at day 0 andRAG−/− OT-I CD45.2 lymphocytes were transferred at day 13: 1 day beforestarting treatment with chemotherapy. The experimental schedule of theGL-261-ova tumor experiments is explained in detail in FIG. 1.

For in vivo cell proliferation tracing, OT-I RAG−/− splenocytes werelabeled with cell proliferation dye eFluor450 (eBioscience, San Diego,Calif.) in the following way: Lymphocytes were isolated from spleencells, their viability was checked post-isolation, and viablelymphocytes were washed with HBSS without Ca²⁺ or Mg²⁺ once inpreparation for the staining. A 10 μM solution of the cell proliferationdye was made in HBSS without Ca²⁺ and Mg²⁺. After washing the isolatedcells, the cells were reconstituted by adding the 10 μM dye solution toa concentration of 5×10⁶ cells/mL. While the solution was added to thecells, the tube was gently vortexed to ensure homogeneous uptake of thedye by all cells.

The cells were incubated for 8 min in the dark at room temperature.Cells were quenched with 20 mls of HBSS with 20% FBS and were then spunat 1400 rpm for 5 min at 4° C. Cells were washed with HBSS without FBSan additional two times and cells were checked for viability. Cells wereresuspended with HBSS (w/o FBS) to a concentration of 10×10⁶ cells/mL.The cells were passed through a 40 μM cell strainer to ensure they werein a single cell suspension and 100 μL of the cell suspension wasinjected retro-orbitally in the recipient mice immediately.

For the adoptive transfer of CD8 cells from the LC+anti-PD1 group to thei.p. BCNU+anti-PD1 group, splenocytes from the LC+anti-PD1 group wereharvested as described before and CD8 cells were isolated by negativeisolation (Dynabeads untouched mouse CD8 cells kit, Life Technologies,Grand Island, N.Y.). Cells were washed with PBS two times afterisolation and were passed through a 40 μM mesh to ensure a single cellsuspension. Cells were resuspended to a concentration of 150×10⁶ CD8cells/mL and 100 μL of the cell suspension was injected in every mousefrom the i.p. BCNU+anti-PD1 rechallenged group via retro-orbitalinjection.

Surgical procedure: polymer implantation: Mice in the LC or EP treatmentgroups were anesthetized with ketamine/xylazine (100 mg/kg ketamine, 10mg/kg xylazine). The skin was prepped with alcohol swabs, the midlineskin incision was opened, and the implantation burr hole created fortumor implantation was identified. The burr hole was re-drilled to allowfor the polymer to enter the cranial cavity. The tumor mass wasidentified and the polymer was pushed directly on top of the tumor andsecured under the skull. The skin incision was closed with staples. Micewere monitored for a period of 30 min after recovery for signs ofneurological deficits.

Anti PD1 monoclonal antibody: Hamster anti-murine PD1 monoclonalantibody-producing hybridoma (G4) was used to produce antibody aspreviously described (Hirano et al., 2005). Hamster immunoglobulinisotype antibody (Rockland Immunochemicals Inc., Gilbertsville, Pa.) wasadministered to animals receiving either BCNU polymers alone or i.p.BCNU alone.

Drugs: BCNU was purchased from Sigma-Aldrich (St. Louis, Mo.) anddissolved in 100% EtOH in preparation for intraperitoneal injection.After completely dissolving BCNU in EtOH, the solution was furtherdiluted with 0.9% saline to a final concentration of 3% EtOH. Mice wereinjected within 5 min of the preparation of the drug.

The construction of BCNU wafers followed the standard protocol (Kim etal., 2007). BCNU was mixed with PCPP-SA polymer powder for a finalconcentration of 3.8% BCNU. EP, used as a control in this study, wasconstructed the same way as the BCNU wafer.

Flow cytometric analysis of tumor infiltrating immune cells andperipheral lymphoid cell populations: At day 21 or day 30 post tumorimplantation, mice were sacrificed using a lethal dose ofketamine/xylazine cocktail. The spleen, brain, cervical lymph nodes(LN), and bone marrow were harvested and passed through a 40-μmstrainer. A 30%-37%-60% Percoll gradient (GE Healthcare,Buckinghamshire, UK) was used to isolate immune cell populations frombrain tumors and the draining LNs. After centrifugation, the 37%-60%interface contained lymphocytes, monocytes, and microglia in the case ofbrain tumors, and lymphocytes and monocytes in the case of draining LNs.

Peripheral blood was collected by cardiac puncture before harvestingother tissues at day 21 and 30. A total of 600 μL was collected fromeach mouse in heparin-coated tubes (BD Biosciences, Franklin Lakes,N.J.). Blood was mixed with PBS in a 1:1 ratio and overlaid onto aFicoll-Paque Plus (GE Healthcare) base in a 1:3 ratio. Aftercentrifugation, peripheral lymphocytes were extracted from the resultinghorizon and were washed twice with PBS.

Lymphocytes from all tissues were stimulated with PMA-Ionomycin andGolgi stop (eBioscience) for 4-6 hours in a 37° C. humidified incubatormaintained at 5% CO₂. For flow cytometric analysis, lymphocytes werestained with CD8 PerCp-Cy5.5 Clone: 53-6.7 (eBioscience), CD3 FITCClone: 17A2 (eBioscience), CD4 APCH7 Clone: GK1.5 (BD Biosciences),FoxP3 PE Clone: MF23 (BD Biosciences), IFN-γ APC Clone: XMG 1.2(eBioscience), and fixable aqua L/D stain (Life Technologies). For thememory T cells, lymphocytes were stained with CD62L PE (BD Biosciences)and CD44 BV421 (BD Biosciences). For the myeloid cells and microglia,the following antibodies were used: Ly-6G APC Clone: RB6-8C5(eBioscience), Ly-6C PE Clone: HK1.4 (eBioscience), CD11b AF700 Clone:M1/70 (Biolegend, San Diego, Calif.), CD11c BV421 Clone: N418(Biolegend), CD45 PE-CF594 Clone: 30-F11 (BD Biosciences). Appropriateisotype controls were used. For the adoptive transfer experiment, CD45.2PE Clone: 104 (eBioscience) was used to stain the isolated lymphocytes.

In addition to the blood draws performed on days 21 or 30 postimplantation, blood draws were also performed on days 26 and 41 postimplantation. On these days, blood was taken via tail vein sampling and100 μL of blood was collected for every mouse. Red blood cells werelysed using RBC Lysis Buffer (eBioscience) and subsequently stained withCD3 FITC Clone: 17A2 (eBioscience). All flow cytometry experiments wereperformed on a LSRII (BD Biosciences) and analysis was performed usingFlowJo software (TreeStar, Ashland, Oreg.).

Re-challenge experiments: Tumor burden was assessed weekly with IVISimaging. Mice were considered “cured” of their brain tumors followinginitial treatment if no evidence of tumor was present at consecutiveIVIS imaging sessions, and mice were further considered long-termsurvivors if no tumor was detected 90 days post implantation. Long-termsurvivors from each experimental group were re-challenged 90 days postimplantation with 300,000 GL-261 LUC cells injected intracranially inthe contralateral hemisphere. Cells were prepared in 14 of PBS. Naïvemice were implanted in parallel as controls, and mice were followed withweekly IVIS imaging.

Statistics: Survival was plotted using Kaplan-Meier curves, and curveswere analyzed with the log-rank Mantel-Cox test using GraphPad Prismsoftware (GraphPad Software, La Jolla, Calif.). For comparison of cellnumbers and percentages between treatment groups in flow cytometryexperiments, a two-tailed unpaired t test was used. P values <0.05 wereconsidered significant.

Example 2 Systemic Chemotherapy is More Immunosuppressive than LocalChemotherapy in the Tumor Microenvironment and the Peripheral LymphoidOrgans

Immediate immunosuppressive effect: To track the evolution of theimmunological response of tumor bearing mice to chemotherapy, the numberof lymphocytes recovered from the brain as well as the spleen, blood,lymph nodes and bone marrow at earlier and later time points along thecourse of chemotherapy was examined (FIG. 1). Mice were followed withserial blood draws, and the blood circulating lymphocytes were trackedby the CD3 surface marker. Mice treated with i.p. BCNU showed adecreased number of CD3+ lymphocytes compared to control mice at the endof the first week of treatment with stably decreasing numbers over thecourse of treatment. At the end of the therapeutic regimen (Day 30 postimplantation), i.p. BCNU treated mice were severely lymphodepleted for along period that lasted for at least 2 weeks after the end of treatment(p-value<0.001) (FIG. 2). The same lymphodepleting pattern was observedin the draining lymph nodes (DLNs). The systemic chemotherapy groupexhibited late myelotoxicity with the overall cellularity of the BM aswell as the leukocytes (as measured by CD45) being lower than thecontrol mice. However, LC did not affect the number of TILs or thelymphocytes in the peripheral lymphoid organs. On the contrary, anincrease in the number of TILs at a late timepoint (Day 30) compared tosystemic BCNU or control mice was observed.

Late immunosuppressive effect: Systemic chemotherapy exhibited aprolonged and potentially irreversible lymphodepletion. A smallpercentage of mice treated with systemic chemotherapy or localchemotherapy were cured of their disease and they were able to befollowed for a period of 4 months from the initial tumor implantation.Mice treated with systemic chemotherapy surprisingly showed a decreasednumber of recovered lymphocytes from the spleen (P=0.002), the LN(P<0.001) and the blood (P=0.07) compared to untreated mice or LCtreated mice (FIG. 3).

Example 3 Use of Systemic Chemotherapy Post Immunotherapy ExhibitsInferior Survival and Immune Activation Compared to Local Chemotherapy

As presented hereinabove, the delivery method of chemotherapy in tumorbearing mice poses a very important effect on the immune system, withsystemic chemotherapy producing severe lymphopenia in the peripherallymphoid organs and the tumor microenvironment while LC preserving anintact immune response. In light of these findings, it was determinedwhether immunotherapy would work best in combination with chemotherapyand if so, which delivery method would maximize the survival benefit andimmune profile of tumor bearing mice. Mice in the EP treatment group hada median survival similar to that of control mice (25 vs 28 days,p=0.1). LC treatment alone increased survival compared to the EP group(28 vs 25 days, p=0.04). An increase in median survival was seen in thei.p. BCNU treatment group compared to LC (28 vs 45 days, p=0.02). Thecombination of i.p. BCNU+anti-PD1 resulted in similar survival comparedto anti-PD1 alone (p=0.6) and showed a trend for increased survivalcompared to BCNU alone (p=0.2). LC+anti-PD1 had the greatest survivalbenefit with LC+anti-PD1 being superior to anti-PD1 alone (p=0.06) or LCalone (p=0.001). More importantly, in a larger cohort of mice (15mice/group), LC+anti-PD1 exhibited higher survival compared to i.p.BCNU+anti-PD1 (p=0.03). FIG. 4 shows the survival data generated incontrol and treated tumor-bearing mice. FIG. 5 shows the tumorprogression of intracranially implanted GL-261 tumors measured bybioluminescent imaging.

Circulating lymphocytes: The anti-PD1 treated group exhibited anincreased number of CD3+ lymphocytes compared to control mice for aprolonged period of time (day 41, FIGS. 6 and 7) after the end of thetherapeutic regimen. LC treated mice showed an increased number of CD3+lymphocytes compared to the control group for an extended period of time(day 41, FIGS. 6 and 7). The combination of i.p. BCNU+anti-PD1 exhibiteda similar lymphodepleting profile as seen after i.p. BCNU treatmentalone: the number of CD3+ blood lymphocytes started decreasing at day 26(FIGS. 6 and 7, p=0.02) and by day 30 reached the same levels as micetreated with i.p. BCNU alone (p=0.9). Mice treated with the combinationof LC+anti-PD1 maintained the high number of blood circulatinglymphocytes compare to the non-treated mice (FIGS. 6 and 7, p=0.5).

Draining lymph nodes: Flow cytometric analysis of the draining lymphnodes confirmed lymphodepletion of both CD3+ cells and CD8+ IFN-γ+ cellsin the i.p. BCNU and i.p. BCNU+anti-PD1 treatment groups. The anti-PD1,LC, and combination LC+anti-PD1 group had higher numbers of CD3+ andhigher percentage of CD8+ IFN-γ+ cells compared to control mice. Thecombination treatment did not result in higher percentage of CD8+ IFN-γ+cells compared to the monotherapies (FIGS. 8 and 9).

Bone marrow (BM) cells: Upon live-dead staining of bone marrow cellsharvested from the femurs of treated and control mice, it was found thatmice in the i.p. BCNU group showed a significant decrease in cellularity(p<0.05). The addition of anti-PD1 failed to reconstitute thecellularity of the BM (p<0.01). The analysis of CD45+ bone marrow cells(leukocytes) from the i.p. BCNU and i.p. BCNU+anti-PD1 groups revealeddecreased cell numbers compared to the control, LC or LC+anti-PD1 groups(p<0.05) (FIG. 10). A more detailed analysis of the specific myeloidsubpopulations can be seen in FIGS. 11 and 12.

Tumor infiltrating lymphocytes (TILs): Mice were sacrificed at days 21and 30 and TILs were analyzed to assess the short-term and long-termeffects of each treatment regimen on the lymphocytes present in thetumor microenvironment. At day 21, on average, 50% of the CD8+ cellsrecovered from control mice produced IFNγ. LC or i.p. BCNU groupsexhibited a similar percentage of CD8+ IFNγ+ cells with 40% and 35% ofthe recovered CD8+ cells producing IFN-γ, respectively (p=0.35 andp=0.5). The addition of anti-PD1 antibody to LC treatment increased thepercentage of CD8-IFNγ producing cells to 70% (p<0.05), which wassimilar to anti-PD1 alone (FIGS. 13 and 14, p=0.87). The percent andnumber of CD4+FoxP3+ regulatory cells was higher in the i.p. BCNU groupcompared to the control (55% vs 30%, p=0.01). LC group moderatelyincreased the percent of Tregs (40%) vs the control or the EP group.Anti-PD1 treatment had the same percentage of Tregs vs control (p=0.96).The addition of anti-PD1 to LC treatment moderately decreased the levelsof CD4+FoxP3+ cells (38%) close to the level of the control mice (FIGS.7 and 8, p<0.001) but did not significantly decrease the percentage ofTregs in combination with i.p. BCNU (50%, p=0.66) (FIGS. 13 and 15).

At day 30 post implantation, the density of CD3+ TILs (number of cellsnormalized by tumor volume) in the i.p. BCNU group was significantlyless than control mice in the control (40 vs 140 cells, p<0.0001). LCenhanced the percentage and number of CD8-IFNγ producing cells (42%, 80cells) compared to the control group (15%, 30 cells, p<0.001).LC+anti-PD1 had a higher percentage of CD8-IFNγ producing cells (38%)compared to the control mice (15%, p<0.0001), but a similar percentagecompared to LC (42%, p=0.38) or anti-PD1 treatment (38%, p=0.75). Thepercent of CD4+FoxP3+ cells was similar at day 30 among the controlgroup (34%), the EP group (33%) and the anti-PD1 (27%) group. LCtreatment continued to have a higher percentage of Tregs (52%) comparedto the control group (27%, p<0.05), but the combination of LC+anti-PD1normalized the percent of CD4+FoxP3+ cells (35%) to that of control mice(P=0.4).

Microglia cells and tumor infiltrating myeloid cells: Resident microgliawere identified as CD11b+CD45[low] cells, infiltratingmacrophages-monocytes as CD11b+CD45[high] cells, and TILs asCD11b-CD45[high] cells. The lymphocytic population was identified asCD11b-CD45[high]. At day 21, the anti-PD1 group and the LC group hadlower levels of activated microglia compared to the control or EP group.The relative percentages of lymphocytes, microglia and monocytesexhibited variability among treatment groups with no distinct patternspecific for any treatment group. The relative percentage of residentmicroglia/macrophages compared to the rest of infiltrating immune cellpopulation strongly correlated with tumor size regardless of thetreatment group at day 21 (p-value=0.003, r²=0.78) (FIGS. 16 and 17).Further analysis of the tumor infiltrating macrophages (TAMs) showed nodistinct pattern among different treatment groups; however, a positivecorrelation existed between tumor size and monocyte/granulocyte ratio(p-value=0.04, r²=0.5). Interestingly, tumor infiltrating dendriticcells defined as CD11b+CD11c+ cells gated from the population of CD45+cells showed an increased % in the LC and LC+anti-PD1 groups compared tothe EP or anti-PD1 treatment (FIG. 18). This result indicates that localchemotherapy is enhancing the infiltration of dendritic cells in thetumor microenvironment that can uptake the released by the dying tumorcells antigens and allow for greater antigen presentation.

Example 4 Use of Systemic Chemotherapy Concurrently with ImmunotherapyExhibits Inferior Survival Compared to Local Chemotherapy and Abrogatesthe Survival Benefit of Immunotherapy

Mice in the LC group trended towards a longer median survival than theEP group (35 vs 23 days, P=0.07). Despite the use of chemotherapy (LC orsystemic chemotherapy) at an earlier timepoint (day 7) (FIG. 19), themedian as well as the long-term survival did not change compared to thelater treatment in the first round of experiments (day 14) (FIG. 4). Theorder of treatment similarly didn't change outcomes as LC prior or afteranti-PD1 administration generated similar survival data (90% vs 80% longterm survivors) (FIG. 4). However, when combining anti-PD1 with i.p.BCNU, systemic chemotherapy abrogated the survival benefit of anti-PD1treatment (30% vs 55% long term survivors) (FIG. 4). Furthermore, thecombination of anti-PD1 and LC showed a statistically significantincrease in survival in comparison to anti-PD1 and systemic chemotherapy(P=0.03).

Example 5 LC Increases the Survival and Homing of Tumor Antigen SpecificT Cells in the Tumor Microenvironment and the Draining Lymph Nodes(DLNs) Whereas Systemic

Chemotherapy is Abrogating this Effect

In light of the flow cytometry results in the GL261 model showing animmune activation with LC and LC and anti-PD1, and to test thehypothesis that this activation occurs as a result ofchemotherapy-induced cell death and subsequent antigen release, theantigen specific ova system was utilized. Mice were implanted withGL-261 ova cells and lymphocytes were adoptively transferred from OT-Imice that are genetically modified to express a T-cell receptor (TCR)with high affinity for the ovalbumin residues 257-264 in the context ofthe H2Kb MHC-I peptide. Four days after the adoptive transfer and threedays after initiation of chemotherapy, the DLNs were harvested as wellas the brains of the mice implanted with G1-261 ova-luc tumors. Flowanalysis with CD3 and CD45.2 markers showed that the LC group had anincreased percentage and number of adoptively transferred ova specific(CD45.2) T cells residing in the DLNs and the brain tumormicroenvironment compared to the systemic chemotherapy groups or theanti-PD1 group (FIGS. 20 and 21). More specifically, a modest increaseof CD3+CD45.2+ in the DLNs was observed in the LC treated mice comparedto anti-PD1 treated mice (8% vs 4%, P=0.3). A significant increase ofCD3+CD45.2+ transferred cells was observed in the LC treated micecompared to systemic BCNU and systemic BCNU and anti-PD1 treated mice(8% vs 3%, P=0.02). These profiles were more exaggerated within thetumor microenvironment; LC treated mice had a significantly higherpercent of CD3+CD45.2+ cells compared to anti-PD1 treatment (18% vs 8%,P=0.02) as well as to i.p.BCNU (18% vs 4%, P=0.012) and i.p. BCNU andanti-PD1 (18% vs 6%, P=0.03) treatment. In vivo cell proliferation ofadoptively transferred lymphocytes migrating to the spleen three daysafter the transfer shows that treatment with LC is allowing for agreater expansion of OT-I cells (94.9% cells divided) compared to EP(47.1%) or No Tx (43.2%) whereas i.p. BCNU is decreasing theproliferation of adoptively transferred lymphocytes (29.9%) (FIG. 22).Without wishing to be bound to any one particular theory, it is believedthat these data support the hypothesis that LC indeed increasesantigen-specific immune activity by increasing antigen-specific immuneactivity.

Example 6 Local Chemotherapy Preserves the Memory Response Against TumorRechallenge whereas Systemic Chemotherapy Abrogates the Creation ofMemory Response and Renders the T Memory Cells Dysfunctional

After establishing that LC can be successfully combined withimmunotherapy and showing that this combination can lead to an increasedtumor specific immune response, it was determined whether theimmunologic response elicited by the combination treatment would exhibita long lasting memory response. In parallel with the LC groups, thememory response of systemic BCNU and systemic BCNU+anti-PD1 treatmentswas assessed.

Mice from the following treatment groups survived over 100 days postinitial tumor implantation with no sign of tumor burden and were thusdeemed long term survivors: anti-PD1, LC, i.p. BCNU, i.p. BCNU+anti-PD1,LC+anti-PD1. In order to assess memory response, long term survivorswere re-challenged with GL-261 cells implanted in the contralateralhemisphere while naïve mice with no previous exposure to tumor cellswere challenged in parallel. No tumor growth occurred in the anti-PD1and LC+anti-PD1 groups indicating a memory response upon tumor antigenrecognition. Naïve mice, in contrast, developed large, progressivelygrowing tumors. Long-term survivors in the i.p. BCNU or i.p.BCNU+anti-PD1 groups were not able to inhibit tumor growth after tumorrechallenge (FIGS. 23 and 24).

Twenty days after intracranial tumor re-challenge, long-term survivormice were assessed for the presence of memory cells.CD3+CD8+CD44[high]CD62L[low] cells were considered to be effector memorycells (T_(EM)) and CD3+CD8+CD44[low]CD62L[high] to be central memory Tcells (T_(CM)). Mice treated with anti-PD1, i.p. BCNU, or LC+anti-PD1exhibited similar percentages of CD4+ or CD8+T_(CM) and T_(EM) cells.Although T_(EM) percentages were similar in all groups, assessment ofIFNγ production after stimulation with PMA/Ionomyocin inCD8+CD44[high]CD62L[low] cells isolated from the DLNs, spleen orperipheral blood revealed dysfunctional IFNγ production in the i.p. BCNUtreatment group as compared to the anti-PD1 or LC+anti-PD1 groups (FIGS.25 and 26).

Example 7 Adoptive Transfer of CD8 Cells from Rechallenged LC+anti-PD1Mice to I.P. BCNU+anti-PD1 Rechallenged Mice Creates Only a PartialAntitumor Response

To identify whether the presence of intact CD8 memory cells could allowi.p. BCNU+anti-PD1 mice to regain their ability to reject the tumorafter tumor rechallenge, CD8 cells were harvested from the spleen ofmice treated with LC+anti-PD1 (that can successfully mount a memoryimmune response) and adoptively transferred in i.p. BCNU+anti-PD1 micerechallenged with the tumor as described in FIG. 27. As mentionedpreviously, mice treated with i.p. BNCU+anti-PD1 and rechallenged withtumor fail to reject the tumor and very quickly grow large tumors fromwhich they die on average at day 16 of the experiment. It is noteworthythat the tumor was very well established when the CD8 cells wereadoptively transferred (day 12). Fifty percent ( 2/4) of the recipientmice showed a progressive decrease in the bioluminescent signal afterthe adoptive transfer of CD8 cells and entered a state of immunologicalequilibrium with the tumor signal being stable for more than three weeks(FIGS. 27 and 28). This indicates that the adoptively transferredeffector CD8 cells were able to slow down the progression of the tumorbut not completely eradicate the recurrent tumors implying that perhapsthe adoptive transfer of memory CD8 cells is not adequate to maintain astrong immune response in these mice. However, when the partialresponders were treated with anti-PD1, they exhibited similar antitumorresponse as chemotherapy naïve mice, indicating that upon infusion andreconstitution of the CD8 cell pool in the systemic chemotherapy mice,anti-PD1 treatment response was corrected (FIG. 28).

Example 8 Recurring Tumors after Tumor Rechallenge in Mice with PriorI.P. BCNU Treatment Cannot be Rescued by Anti-PD1 Treatment

In a separate set of experiments, long-term survivor mice from thesystemic chemotherapy group were rechallenged with tumor and weretreated with anti-PD1 in an attempt to salvage these mice from tumorprogression. Mice implanted with primary tumor and treated with anti-PD1only exhibited a 50% long term survival as expected, whereas micetreated with systemic chemotherapy for their primary tumor and salvagedwith anti-PD1 for their rechallenged tumor (i.p. BCNU R and rescueanti-PD1) failed to respond to anti-PD1 treatment (FIG. 29);additionally, the rate of tumor progression in the i.p. BCNU R andrescue anti-PD1 group, as measured by bioluminescent imaging, wassimilar to the rate of tumor bearing mice that did not receive anytreatment (FIG. 29). After harvesting peripheral blood, spleen and thebrain of these rechallenged mice, it was confirmed that T memory cellsfrom the systemic chemotherapy+anti-PD1 treatment mice exhibited adysfunctional IFNγ production (FIGS. 30 and 31) compared to the LC andanti-PD1 treated mice, but it was further observed that anti-PD1administration to the BCNU R mice did not restore the functionality oftheir memory T cells. More specifically, T memory cells in the brainparenchyma of LC and anti-PD1 treated mice showed a dramatically higherIFNγ production compared to i.p. BCNU R and rescue anti-PD1 mice (70% vs20%, P=0.02). The same patterns of IFNγ production from T memory cellswere observed in the peripheral blood with LC and anti-PD1 exhibitingthe highest T memory associated IFNγ production (FIGS. 30 and 31). Uponnecropsy of these mice at the experimental day 104, the size of thespleen in the systemic chemotherapy treated mice was significantlydecreased compared to the LC and anti-PD1 treated mice (FIG. 32).

Example 9 Discussion

The use of chemotherapy in combination with immunotherapy hasundoubtedly complex interactions. The doses used, the delivery method,as well as the type of chemotherapy and immunotherapy can affect thefinal outcome (Jackson et al., 2013). Chemotherapies with mildimmunosuppressive effects, such as temozolamide, could allow for apotential therapeutic window of use with immunotherapy (van der Most etal., 2005). Certain chemotherapies seem to have differential effects inspecific lymphoid subsets, such as cyclophosphamide, whichpreferentially depletes Tregs in low doses (Walter et al., 2013; Le andJaffee, 2012). The type of cell death may also be important instimulating the immune system. It has been hypothesized that cytotoxictreatments that lead to apoptosis are less effective in inducing arobust immune response as the organized cell death (apoptosis) does notallow for tumor antigen presentation, as compared to the antigen releasethat occurs with necrosis (van der Most et al., 2005). The timing ofchemotherapy in combination with immunotherapy is also critical. Thedata suggest that administering immunotherapy to patients who havepreviously received systemic chemotherapy may render theimmunotherapeutic agent ineffective.

The GL-261 syngeneic mouse glioblastoma model was used to assess theefficacy of combining PD1 blocking antibody with systemic or local BCNU.In a series of experiments, the different effects of local vs. systemicBCNU were defined on the cellular anti-tumor immune response andsurvival experiments were conducted to determine if the observedimmunologic advantage translated to prolonged survival. It was foundthat local, rather than systemic, delivery of BCNU in combination withPD1 blockade is immunologically superior and results in significantlylonger survival. Systemic chemotherapy depleted the lymphocytepopulation in the tumor mass, peripheral lymphoid organs, and peripheralblood. Administration of systemic BCNU to anti-PD1 treated miceabrogated the characteristic immunologic profile of anti-PD1 therapy.Conversely, the combination of local chemotherapy (LC) and PD1 blockadewas associated with a robust immune response and increased survivalcompared with either monotherapy. These findings were validated in anantigen specific in vivo system; OT-I T cells were adoptivelytransferred to mice bearing GL-261-ova tumors undergoing treatment witha combination of chemotherapy and/or anti-PD1.

The series of experiments described hereinabove serve as evidence thatlocally delivered chemotherapy may be combined successfully withanti-PD1 mAb. The results indicate that biodegradable BCNU wafers incombination with anti-PD1 yield superior survival and immune stimulationcompared to the combination of systemic, intraperitoneal BCNU (i.p.BCNU) and anti-PD1. Mechanistically, this treatment regimen affectsseveral cell populations, including T cells, dendritic cells, migratingmyeloid cells, and local microglia. Furthermore, in studying the effectLC or LC and anti-PD1 has on antitumor antigen specific T cellresponses, it is shown that OT-I T cells adoptively transferred to micebearing GL-261-ova tumors exhibit higher clonal expansion at both thedraining lymph nodes as well as in the tumor microenvironment comparedto all other treatment groups. These results imply that LC stimulatestumor-directed T cell responses triggered by increased antigen releasefrom chemotherapy induced cell death. Although local BCNU+anti-PD1generated the most long-term survivors, a percentage of mice from eachtreatment group became long-term survivors in this model. Upon tumorre-challenge, however, mice previously treated with i.p. BCNU+anti-PD1did not reject brain tumor formation, whereas mice treated withLC+anti-PD1 rejected tumor formation. Furthermore, when the BCNUrechallenged mice were attempted to be salvaged with anti-PD1 treatment,the mice did not respond to treatment unlike the control mice treatedwith anti-PD1 that exhibited a complete response in 50% of the animals.

Accordingly, the presently disclosed subject matter demonstrates thatlocal chemotherapy in the form of controlled-release BCNU polymers issuperior to systemic BCNU for combination with anti-PD1 immunotherapy.This regimen provides a robust survival benefit as well as an increasein tumor-infiltrating immune cells and formation of memory cellsnecessary to resist tumor re-challenge. Taken together, these resultshighlight the importance of rigorously testing the effects of order,timing, and dosage in implementing combinationchemotherapy/immunotherapy regimens.

While chemotherapy and immunotherapy have been widely used and validatedindependently, the combination of these two modalities has thus far beendiscouraged due to the known effects of chemotherapy on the immunesystem (van der Most et al., 2005). It is hypothesized thatadministration of LC would not have the same systemic immunosuppressiveeffects as systemic therapy and, therefore, is a preferable strategy forcombination with immunotherapy (Jackson et al., 2013). In fact, it wasfound that local BCNU not only allowed for retained activity ofanti-PD1, but actually boosted the immune response. One potentialmechanism for this synergy might be increased antigen presentation astumor cells die in response to chemotherapy. This hypothesis issupported by the increased percentage of DCs present in the LC groupscompared to the control group or the anti-PD1 group. Tumor infiltratingdendritic cells (TIDC) have been shown to be potent antigen-presentingcells (Preynat-Seauve et al., 2006). By implanting GL-261 ova expressingcells in mice and adoptively transferring OT-I lymphocytes, it wasconfirmed that the increased TIDC in the LC allowed for a greaterexpansion of the adoptively transferred ova-specific lymphocytes in thebrain and the DLNs compared to mice treated with anti-PD1 alone oranti-PD1 in combination with systemic BCNU. These results support thehypothesis that chemotherapy induced cell death as a result of LCtreatment attracts more DCs, which uptake the released antigens allowingfor a greater antigen presentation and further clonal activation oftumor specific T cell responses. However, the use of systemicchemotherapy seems to abrogate the creation of the antitumor antigenspecific T cell responses.

These immunologic findings are consistent with the survival data. LC incombination with anti-PD1 exhibited the most robust survival benefitcompared to the control and monotherapy groups, indicating a synergisticrelationship leading to durable anti-tumor responses. Furthermore, thesedata show that systemic BCNU treatment (i.p. BCNU) does not worksynergistically with anti-PD1. Interestingly, in this model, it wasobserved that anti-PD1 treatment had a very quick anti-tumor responsewith 50% of the mice that ended up being long-term survivors losingtheir BLI signal as early as day 14 (at the end of anti-PD1 treatmentand the beginning of BCNU treatment). This implies that long-termsurvivorship in the i.p. BCNU+anti-PD1 group was mainly due to theanti-PD1 effect rather than the systemic BNCU administration. The use ofsystemic chemotherapy alone or in combination with anti-PD1 resulted ina delay in tumor progression compared to control or anti-PD1 treatmentrespectively; however the tumors gradually grew back resulting in latemortality, consistent with the results in human glioblastoma treatment(Stupp et al., 2009). Consistent with this argument are the results ofthe survival experiment where systemic chemotherapy was given prior toimmunotherapy; addition of systemic chemotherapy to anti-PD1 did notprovide a survival benefit and was in fact inferior to PD1 monotherapy(FIG. 19). Taken together, these data indicate that local and notsystemic chemotherapy generates a significant increase in survival whencombined with PD1 blockade over either monotherapy.

The effective combination of LC and anti-PD1 resulted in an enhancedanti-tumor immune response within the tumor microenvironment, furthersupporting the utility of combining chemotherapy and immunotherapy. Theimmunologic profile exhibited by mice treated with LC+anti-PD1 showed adecreased number of CD4+FoxP3+ cells and an increased number of CD8+IFNγ producing cells as compared to mice in monotherapy or controlgroups. Interestingly, immune infiltration of CD8+ IFNγ producing cellswas seen at day 30, but not at day 21, which could point to the localcytoreductive effect of BCNU treatment giving way to immune activation.The interaction between LC and anti-PD1 treatment and its effect onimmune infiltration likely occurs on two fronts: the cytoreductiveeffect of the BCNU is concentrated within the first week after polymerimplantation from day 14 to day 21 post tumor implantation due to thekinetics of BCNU release from the polymer (Fleming and Saltzman). Thepresence of BCNU in the tumor microenvironment leads to cell death anddecreased proliferation of immune cells infiltrating the tumormicroenvironment at day 21 as seen with other locally administeredchemotherapy (Litterman, Dudek, et al., 2013; Litterman, Zellmer, etal., 2013). Persistent antigen stimulation, as a result ofchemotherapy-induced tumor cell death at the first week of polymerimplantation continues to attract effector T cells in the tumor; thedecreased concentration of BCNU the second week after polymerimplantation allows for effector T cells to exert their anti-tumorfunction (Hailemichael and Overwijk). Both local chemotherapy andsystemic chemotherapy increased the percent of T-regulatory cells at day21 and day 30. The addition of anti-PD1 to local chemotherapy but not tosystemic chemotherapy decreased the percentage of T-regulatory cells atdays 21 and 30 leading to an increased Teff/Treg ratio in theLC+anti-PD1 group at day 30. LC and anti-PD1 monotherapy groups wereincluded in the flow cytometric analysis to identify the contributioneach treatment had in the tumor microenvironment. As expected, theanti-PD1 monotherapy group exhibited increased infiltration of TILs,with increased numbers of CD8+ IFNγ producing cells and decreasednumbers of CD4+FoxP3+ cells (Pardoll, 2012) whereas LC increased theCD4+FoxP3+ cells and provided an initial (day 21) moderate decrease ofCD8+ IFNγ producing cells. However, LC enriched the tumormicroenvironment for CD8+ IFNγ producing cells at day 30 compared to thecontrol group.

Further, the effect of chemotherapy and/or anti-PD1 on peri-tumoralmicroglia was explored. As described previously (Gabrusiewicz et al.,2011), resident microglia promote an immunosuppressive microenvironmentfacilitating the establishment and progression of GBM. The interactionbetween glial cells and microglia transforms the latter to the ameboid(activated) state and selectively activates the MAPK pathway without thesecretion of pro-inflammatory cytokines. The release of TGF-b andseveral other immunosuppressive cytokines from activated microglia incombination with dysfunctional Toll-like receptor (TLR) responsesprevents microglia from functioning as scavenger-antigen presentingcells (APCs), as is the case in the tumor-free state (Hussain et al.,2006). Consistent with previous studies, the percent of residentmicroglia/macrophages (CD11b+, CD45+) in control mice post tumorimplantation constitutes the majority of glioma infiltrating immunecells. However, anti-PD1 treatment preferentially increases the percentof TILs compared to resident microglia/macrophages. This hypothesis issupported by an increased Teff/Treg ratio, favoring an effectorphenotype in the anti-PD1 group. LC increased the percent of both TILsand microglia leading to a higher percent of microglia compared to TILs.This observation warrants further investigation as the positive TILs:microglia/macrophages ratio may be an artifact of reduced tumor size.This hypothesis is supported by the strong correlation between tumorsize and microglia/macrophage: TILs ratio (p-value=0.003, r²=0.78) andthe absence of a distinct pattern in the above ratio when comparingdifferent treatment groups.

The immune profile in the peripheral blood and lymphoid organs reflectedthe changes depicted in the tumor microenvironment. Systemicadministration of BCNU depleted CD3+ cells in the draining LNs, theperipheral blood and caused myelotoxicity. CD8+ IFNγ-producing cellswere increased by percentage in both the peripheral blood and thedraining lymph nodes in the combination LC+anti-PD1 group. LC oranti-PD1 alone did not have any effect on the percent of CD8+ cellsproducing IFNγ producing cells in the periphery. Although the systemicadministration of BCNU did not affect the percentage of CD8+ IFNγproducing cells, it did greatly deplete the number of cells present inthe peripheral organs.

The re-challenge experiments show that anti-PD1 therapy generatesimmunologic memory against tumor antigens, resulting in tumor rejectionupon re-challenge. Interestingly, systemic BCNU abrogated this effect.Local BCNU, however, allowed for persistence of immunologic memoryimparted with anti-PD1 therapy. Flow cytometric analysis ofre-challenged mice supports the hypothesis that i.p. BCNU disruptsmemory T cell function. The analysis of T_(EM) cells from the peripherallymphoid organs (DLNs, peripheral blood, spleen) shows that cells fromi.p. BCNU-treated mice exhibit dysfunctional IFNγ production comparedwith anti-PD1 or LC+anti-PD1 groups. Furthermore, the lack of a completeantitumor response in i.p. BCNU+anti-PD1 rechallenged mice afteradoptive transfer of CD8 cells from LC+anti-PD1 mice that rejected thetumor upon rechallenge implies that CD8 memory cells alone are notsufficient to produce a rapid and complete antitumor response. Withoutwishing to be bound to any one particular theory and putting thesurvival data and the rechallenge-memory response results together, itcan be postulated that the late recurrence of the disease after systemicBCNU treatment (as shown in the survival experiments, but moreimportantly in the treatment responses in patients) may be attributed totwo factors: a) acquired mutations of the tumor after the use ofDNA-damaging agents and b) the lack of effector memory cells that caneliminate tumor clones encountered in the past but not successfullyeliminated at that time. Additionally, i.p. BCNU R and rescue anti-PD1mice did not show a survival or immunologic response to anti-PD1treatment as expected in chemotherapy naïve anti-PD1 treated mice.

Conclusively, the presently disclosed subject matter provides evidenceof superior immunological response and greater tumor regression when LCand anti-PD1 treatment are combined. Systemic BCNU abrogates thepositive survival and immunological profile anti-PD1 provides to micebearing murine glioblastoma tumors. Furthermore, systemic chemotherapy,unlike LC, fails to prevent recurrence of the tumor upon tumorrechallenge and abrogates the memory response created by anti-PD1. Thelack of memory response upon tumor rechallenge can be explained by adecrease in the memory T cell response.

Accordingly, it is concluded that intratumoral, controlled release BCNUis a superior treatment modality compared with systemic BCNU forcombination with immunotherapy. This strategy circumvents theimmunosuppressive effects of systemic chemotherapy and has aparticularly robust effect on maintaining the memory T-cell population.Since BCNU-eluting polymers are currently approved for use in recurrentand newly diagnosed glioblastoma, this strategy may be readilytranslated into clinical trials. Immunotherapy and anti-PD1 mAbspecifically has been successfully used for the treatment of non-centralnervous system tumors but, based on the results presented herein, itstherapeutic effect might be underestimated due to its use post systemicchemotherapy. The results presented here can have significantimplications on the therapeutic strategy used for the treatment ofmultiple cancer types, such as glioblastoma.

REFERENCES

All publications, patent applications, patents, and other referencesmentioned in the specification are indicative of the level of thoseskilled in the art to which the presently disclosed subject matterpertains. All publications, patent applications, patents, and otherreferences are herein incorporated by reference to the same extent as ifeach individual publication, patent application, patent, and otherreference was specifically and individually indicated to be incorporatedby reference. It will be understood that, although a number of patentapplications, patents, and other references are referred to herein, suchreference does not constitute an admission that any of these documentsforms part of the common general knowledge in the art. In case of aconflict between the specification and any of the incorporatedreferences, the specification (including any amendments thereof, whichmay be based on an incorporated reference), shall control. Standardart-accepted meanings of terms are used herein unless indicatedotherwise. Standard abbreviations for various terms are used herein.

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Although the foregoing subject matter has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be understood by those skilled in the art thatcertain changes and modifications can be practiced within the scope ofthe appended claims.

1-22. (canceled)
 23. A method for treating human glioblastoma andreducing glioblastoma recurrence in humans, the method consisting of:(i) surgically removing all or a portion of a solid glioblastoma tumorin a human patient with recurrent glioblastoma multiforme; and (ii)administering to the human patient with recurrent glioblastomamultiforme an effective amount of a combination treatment consisting of:(a) a locally administered chemotherapy comprising a BCNU implantablewafer; and (b) lambrolizumab, wherein recurrence of the glioblastoma isinhibited.
 24. The method of claim 23, wherein locally administeredchemotherapy is administered intratumorally and/or within a tumor bed.25. The method of claim 23, wherein CD8+ IFNγ producing cells areincreased in the human patient with recurrent glioblastoma multiforme.26. The method of claim 23, wherein the locally administeredchemotherapy increases antigen release from tumor cells in the humanpatient with recurrent glioblastoma multiforme.
 27. A method forreducing recurrence of recurrent human glioblastoma multiforme, themethod consisting of: administering to a human patient with recurrentglioblastoma multiforme an effective amount of a combination treatmentconsisting of: (a) a locally administered chemotherapy comprising a BCNUimplantable wafer; and (b) lambrolizumab, wherein recurrence of theglioblastoma multiforme is inhibited.
 28. The method of claim 27,wherein locally administered chemotherapy is administered intratumorallyand/or within a tumor bed.
 29. The method of claim 27, wherein CD8+ IFNγproducing cells are increased in the human patient with recurrentglioblastoma multiforme.
 30. The method of claim 27, wherein the locallyadministered chemotherapy increases antigen release from tumor cells inthe human patient with recurrent glioblastoma multiforme.
 31. A methodfor increasing antigen release from tumor cells in a human patient withglioblastoma, the method consisting of: administering to the humanpatient with glioblastoma an effective amount of a combination treatmentconsisting of: (a) a locally administered chemotherapy comprising a BCNUimplantable wafer; and (b) lambrolizumab.
 32. The method of claim 31,wherein the glioblastoma is recurrent glioblastoma multiforme.
 33. Amethod for increasing CD8+ IFNγ producing cells in a human patient withglioblastoma, the method consisting of: administering to the humanpatient with glioblastoma an effective amount of a combination treatmentconsisting of: (a) a locally administered chemotherapy comprising a BCNUimplantable wafer; and (b) lambrolizumab.
 34. The method of claim 33,wherein the glioblastoma is recurrent glioblastoma multiforme.